Branched-chain amines in electrochemiluminescence detection

ABSTRACT

The disclosure concerns methods for the detection of an analyte in a sample by electro-chemiluminescence using new reagent compositions. New reagent compositions, reagent kits for measuring electrochemiluminscence (ECL) and electrochemiluminescence detection methods using the new reagent compositions are disclosed. In particular, the disclosure relates to the use of novel combinations of compounds which can be used in said measurements to provide improved assay performance.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/EP2017/055671 filed Mar. 10, 2017, which claims priority to EuropeanApplication No. 16159835.4 filed Mar. 11, 2016, the disclosures of whichare hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention concerns methods for the detection of an analyte in asample by electrochemiluminescence using new reagent compositions. Newreagent compositions, reagent kits for measuring electrochemiluminscence(ECL) and electrochemiluminescence detection methods using the newreagent compositions are disclosed. In particular, the invention relatesto the use of novel combinations of compounds which can be used in saidmeasurements to provide improved assay performance.

BACKGROUND

Methods for measuring electrochemiluminescent phenomena have been knownfor some years. Such methods make use of the ability of special metalcomplexes to achieve, by means of oxidation and reduction reactions, anexcited state from which they decay to ground state, emitting photons.For review see Richter, M. M., Chem. Rev. 104 (2004) 3003-3036.

At this time, there are a number of commercially available instrumentsthat utilize electrochemiluminescence (ECL) for analytical measurements,e.g. in the field of in vitro diagnostic application. Species that canbe induced to emit ECL (ECL-active species) have been used as ECLlabels. Examples of ECL labels include organometallic compounds such asthe tris-bipyridyl-ruthenium (RuBpy) moiety where the metal is from, forexample, the metals of group VII and VIII, including Re, Ru, Ir and Os.Species that react with the ECL label in the ECL process are referred toherein as ECL coreactants. Commonly used coreactants for ECL includetertiary amines (e.g. tripropylamine (TPA)), oxalate, and persulfate.The light is generated by ECL labels or ECL ligands; the participationof the binding reagent in a binding interaction can be monitored bymeasuring ECL emitted from the ECL label. Alternatively, the ECL signalfrom an ECL-active compound may be indicative of the chemicalenvironment (see, e.g., U.S. Pat. Nos. 5,641,623 and 5,643,713, whichdescribes ECL assays that monitor the presence or destruction of specialECL coreactants). For more background on ECL, ECL labels, ECL assays andinstrumentation for conducting ECL assays see U.S. Pat. Nos. 5,093,268;5,147,806; 5,240,863; 5,308,754; 5,324,457; 5,591,581; 5,597,910;5,679,519; 5,705,402; 5,731,147; 5,786,141; 5,846,485; 5,866,434;6,066,448; 6,136,268 and 6,207,369, and EP 0 441 875, and published PCTNos. WO90/05296, WO97/36931; WO98/12539; WO99/14599; WO99/32662;WO99/58962; WO99/63347; WO00/03233 and WO98/57154.

Commercially available ECL instruments for in vitro diagnostics havedemonstrated exceptional performance. They have become widely used forreasons including their excellent sensitivity, dynamic range, precision,and tolerance of complex sample matrices. The commercially availableinstrumentation uses flow cell-based designs with permanent reusableflow cells.

Available sample volumes for the determination of analytes are oftenlimited and more and more different analytes have to be determined outof one sample. Also the development of faster laboratory equipment forassay automation and more sensitive methods for the detection ofanalytes are required. This leads to the need for highly sensitive andspecific electrochemiluminescent assays and methods for performing them.In addition improvements associated with safety hazards or environmentalconcerns should be considered.

In particular, still more sensitive detection of analytes would be ofgreat advantage. Thus, the object of the present invention was toimprove said known methods and reagent compositions especially withrespect to enhancement of the ECL signal and an improved analytedetection in combination with electrochemiluminescent procedures. Itwould be desirable to find novel signal enhancing reagents and/orcompounds with improved performance in electrochemiluminescent assays.

SUMMARY OF THE INVENTION

In an embodiment, the present invention relates to a method of detectingan electrochemiluminescence (ECL) signal comprising

a) contacting a reaction composition comprising

-   -   i) at least one branched-chain tertiary amine and    -   ii) an ECL compound comprising a transition metal complex with        an electrode,

b) electrochemically triggering the release of luminescence, and

c) detecting the ECL signal.

The present invention, in a further embodiment, concerns a method fordetecting an analyte in a sample via electrochemiluminescence detection,comprising the steps of:

a) incubating the sample with a detection reagent labeled with anelectrochemiluminescent group comprising a transition metal complex, inan embodiment comprising a tris(2,2′-bipyridyl)ruthenium complex(Ru(bpy)₃ ²⁺),

b) separating analyte-bound and analyte-free labeled detection reagent,

c) contacting the separated analyte-bound labeled detection reagent witha branched-chain tertiary amine of the present invention and with anelectrode,

d) electrochemically triggering the release of luminescence, and

e) detecting the electrochemiluminescence (ECL) signal thereby detectingthe analyte.

In a further embodiment, the present invention relates to a reagentcomposition for detecting ECL, comprising

i) a branched-chain tertiary amine, in particular a branched-chaintertiary amine of Formula I

as coreactant, and

ii) a further ECL reagent.

In an embodiment, the present invention also relates to an ECL reactioncomposition comprising i) at least one ECL compound comprising atransition metal complex and ii) at least one branched-chain tertiaryamine as a coreactant.

The present invention also relates to a use of a branched-chain tertiaryamine as specified in any one of the present invention, of a reagentcomposition according to the present invention, and/or of reactioncomposition according to the present invention in the detection of ECL.

Further, the present invention relates to a kit for detecting ECLcomprising i) a branched-chain tertiary amine and ii) an ECL reagent.

Moreover, the present invention relates to an ECL device comprising abranched-chain tertiary amine as specified in any one of embodiments ofthe present invention.

Furthermore, the present invention relates to an ECL device comprising abranched-chain tertiary amine of the present invention.

The invention, as well as additional objects, features and advantagesthereof, will be understood more fully from the following detaileddescription of certain embodiments.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the ECL signal measured in dependence on the appliedmeasuring voltage in an artificial immunoassay including RuBpy labeledmicroparticles (“SAP assay”) for 6 different coreactands. X-Axis:measuring voltage in mV, Y-Axis: signal intensity/maximal signalintensity (S/Smax); TPA: Tripropylamine, TBA: Tributylamine, EP:Ethylpiperidine, DIEA: N,N-Diisopropyl-N-ethylamine, DPIBA:N,N-Dipropyl-N-(sec-butyl)amine, DIBPA: N,N-Di(sec-butyl)-propylamine.

FIG. 2 shows the ECL signal measured in dependence on the appliedmeasuring voltage in the absence of RuBpy labeled microparticles(“background signal”) for 6 different coreactands. X-Axis: measuringvoltage in mV, Y-Axis: signal intensity/maximal signal intensity(S/Smax); for further abbreviations cf. legend to FIG. 1 .

FIG. 3 shows the signal to background ratio (S/BG) in dependence on theapplied measuring voltage for 4 different coreactands. X-Axis: measuringvoltage in mV, Y-Axis: signal to background ratio.

FIG. 4 comparison of background signal and positive control signals withvarious beads and two different coreactands in the SAP assay. Relativebackground (“low”) and positive control signals (“high”) for variousbead types and buffers containing DBIPA or TPA. The signal obtained withTPA was set to 1. Apparently the usage of DPIBA leads to a decrease inbackground (low) and specific (high) signals. The decrease in thebackground signal however is stronger compared to the decrease inspecific signal. This leads to an improved overall S/BG ratio.

FIG. 5 improvement of S/BG ratio; S/BG ratio of samples measured in thepresence of DPIBA relative to sample measured with TPA.

FIG. 6 comparison of Ru and Ir as transition metals in the method of thepresent invention in the SAP assay. If Ir label is used the decrease inthe specific signal upon measuring at reduced voltage is lower comparedto Ru label.

FIG. 7 shows the cyclic voltammetry data for 3 different coreactands.X-Axis: applied voltage in V, Y-Axis: current in mA; TPA:Tripropylamine, DPIBA: N,N-Dipropyl-N-(sec-butyl)amine, DIBPAN,N-Di(sec-butyl)-propylamine. With increasing number of branchedsubstituents the oxidation current at a given potential increases due toa decrease of the standard oxidation potential of the compound.

DETAILED DESCRIPTION

The practicing of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology(including recombinant techniques), microbiology, cell biology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook etaI., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “AnimalCell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology”(Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M.Ausubel et al., eds., 1987, and periodic updates); “PCR: The PolymeraseChain Reaction”, (Mullis et al., eds., 1994).

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology, 2^(nd) ed., J. Wiley & Sons, NewYork (1994); March, Advanced Organic Chemistry Reactions, Mechanisms andStructure, 4th ed., John Wiley & Sons, New York (1992); Lewin, B., GenesV, published by Oxford University Press (1994), ISBN 0-19-854287 9);Kendrew, J. et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd. (1994), ISBN 0-632-02182-9); andMeyers, R. A. (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc. (1995),ISBN 1-56081-569 8) provide one skilled in the art with a generalguidance to many of the terms used in the present application.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

As used herein, each of the following terms has the meaning associatedwith it in this section.

As used in the following, the terms “have”, “comprise” or “include” orany arbitrary grammatical variations thereof are used in a non-exclusiveway. Thus, these terms may both refer to a situation in which, besidesthe feature introduced by these terms, no further features are presentin the entity described in this context and to a situation in which oneor more further features are present. As an example, the expressions “Ahas B”, “A comprises B” and “A includes B” may both refer to a situationin which, besides B, no other element is present in A (i.e. a situationin which a solely and exclusively consists of B) and to a situation inwhich, besides B, one or more further elements are present in entity A,such as element C, elements C and D or even further elements.

Further, as used in the following, the terms “preferably”, “morepreferably”, “most preferably”, “particularly”, “more particularly”,“specifically”, “more specifically” or similar terms are used inconjunction with optional features, without restricting furtherpossibilities. Thus, features introduced by these terms are optionalfeatures and are not intended to restrict the scope of the claims in anyway. The invention may, as the skilled person will recognize, beperformed by using alternative features. Similarly, features introducedby “in an embodiment of the invention” or similar expressions areintended to be optional features, without any restriction regardingfurther embodiments of the invention, without any restrictions regardingthe scope of the invention and without any restriction regarding thepossibility of combining the features introduced in such way with otheroptional or non-optional features of the invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an analyte” means one analyte or more thanone analyte. The term “at least” is used to indicate that optionally oneor more further objects may be present. By way of example, an arraycomprising at least two discrete areas may optionally comprise two ormore discrete test areas. The expression “one or more”, in anembodiment, denotes 1 to 50, in a further embodiment 1 to 20, in afurther embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15. Moreover,if not noted otherwise, the term “about” relates to the indicatedvalue±20%.

The methods of the present invention, in an embodiment, are in vitromethods. Moreover, they may comprise steps in addition to thoseexplicitly mentioned herein. For example, further steps may relate,e.g., to obtaining a sample for analysis, or calculating a measurementvalue or a corrected measurement value from an ECL value detected.Moreover, one or more of the steps of the methods of the presentinvention may be performed by automated equipment.

“Detection” includes any method of detecting, including direct andindirect detection. The term “detection” is used in the broadest senseto include both qualitative and quantitative measurements of anelectrochemiluminescence signal; and qualitative and quantitativemeasurements of an analyte, herein also referred to as measurements ofan analyte. In one aspect, a detection method as described herein isused to identify the mere presence of ECL in a detection composition orof an analyte of interest in a sample. In another aspect, the method canbe used to quantify an ECL signal in a detection composition or anamount of analyte in a sample. For quantitative detection, either theabsolute or precise intensity of an ECL signal or amount of an analytewill be detected; or the relative intensity of an ECL signal or amountof an analyte will be detected. The relative intensity or amount may bedetected in a case where the precise intensity or amount can or shallnot be detected. In said case, it can be detected whether the intensityor amount is increased or diminished with respect to a second sampleproviding a reference intensity or comprising said analyte in a second,in an embodiment pre-determined, amount.

In an embodiment, detecting is measuring. The terms“measuring”/“measurement” in science relate to the process of estimatingor determining the magnitude of a quantity, such as length or mass,relative to a unit of measurement, such as a meter or a kilogram.Measuring/measurement uses a reference point against which other thingscan be evaluated. The term measurement can also be used to refer to aspecific result (determined values) obtained from a measurement process.It is a basis for comparison. The skilled artisan is aware of materialsand methods to correlate measured signals or determined values toconcentrations.

To “reduce” or “inhibit” is to decrease or reduce an activity, function,and/or amount as compared to a reference. By reduce or inhibit is meantthe ability to cause an overall decrease in an embodiment of 10% orgreater, in a further embodiment of 25% or greater, in a furtherembodiment of 50%, 75%, 90%, 95%, or greater.

To “enhance”, e.g. to “enhance specific signals” or “the enhancement ofECL signals”, is to increase or rise an activity, function, and/oramount as compared to a reference. By increase or rise is meant theability to cause an overall increase in an embodiment of 10% or greater,in a further embodiment of 25% or greater, in a further embodiment of50% or greater.

The term “luminescence” refers to any emission of light that does notderive energy from the temperature of an energy source (for example, asource of electromagnetic radiation, a chemical reaction, mechanicalenergy). In general, the source causes an electron of an atom to movefrom a lower energy state into an “excited” higher energy state; thenthe electron releases that energy in the form of emitted light when itfalls back to a lower energy state. Such emission of light usuallyoccurs in the visible or near-visible range of the electromagneticspectrum. The term “luminescence” includes, but is not limited to, suchlight emission phenomena such as phosphorescence, fluorescence,bioluminescence, radioluminescence, electroluminescence,electrochemiluminescence and thermo-luminescence. In an embodiment,luminescence is electrochemiluminescence (ECL). As is understood by theskilled person, ECL is luminescence produced during an electrochemicalreaction as specified elsewhere herein. Accordingly, an ECL signal, inan embodiment, is a detectable electrochemiluminescent signal, whethervisibly detectable or detectable by using suitable instrumentation, inan embodiment photometric instrumentation.

The term “contacting”, as used in the context of the methods of thepresent invention, is understood by the skilled person. In anembodiment, the term relates to bringing a compound into physicalcontact with a further compound or device, thereby allowing the compoundand the further compound or device to interact. In particular the termrelates to bringing a branched-chain tertiary amine of the presentinvention into physical contact with an ECL compound comprising atransition metal complex and an electrode; and/or to bringing adetection reagent into physical contact with a sample.

The term “transition metal complex”, as used herein, relates to acompound comprising a transition metal ion complexed by an appropriatecomplexing agent. The term “compound comprising a transition metalcomplex” relates to any compound comprising a transition metal complexand a second chemical compound. In an embodiment, the transition metalcomplex and the second chemical compound are linked covalently. In afurther embodiment, the second chemical compound is a biologicalmacromolecule. In further embodiment, the second chemical compound is ananalyte specific reagent as specified below. As used herein, the term“ECL compound comprising a transition metal complex” relates to acompound comprising a transition metal complex wherein the transitionmetal complex emits ECL under appropriate conditions. In an embodiment,the ECL compound is comprising a transition metal complex. In anembodiment, the transition metal is selected from the group consistingof Ruthenium (═Ru), Iridium (═Ir), Rhenium, Osmium, Europium, Terbium,and Dysprosium; in a further embodiment, the transition metal isRuthenium, Iridium, Rhenium, or Osmium; in a further embodiment, thetransition metal is Ruthenium or Iridium. Appropriate complexing agentsare known in the art and include bipyridine, phenanthroline,phenyl-pyridine, phenyl-quinoline, phenylphenanthridine orpyridine2-carboxylic acid.

ECL compounds comprising a transitional metal complex are for exampledisclosed in WO 8706706 A1, WO 2003002974, EP720614(A1) and U.S. Pat.No. 6,451,225 (B1).

In an embodiment, the ECL compound comprising a transition metal complexis selected from the group consisting of

Ru(bpy)2-bpyCO—OSu, which is the N-hydroxy-succinimide ester of CAS Reg.Nr.115239-59-3 (Ru(bpy)2-bpyCO2H)═BPRu, also known in the art asRuthenium(1+),bis(2,2′-bipyridine-κN1,κN1′)(4′-methyl[2,2′-bipyridine]-4-butanoato-κN1,κN1′)-,(OC-6-33)-, hydrogen hexafluorophosphate(1−) (1:1:2), also known asRuthenium(1+),bis(2,2′-bipyridine-N,N′)(4′-methyl[2,2′-bipyridine]-4-butanoato-N1,N1′)-,(OC-6-33)-, hydrogen hexafluoropho sphate(1−) (1:1:2));

Sulfo-BPRu NHS Ester (=CAS Reg. Number 482618-42-8 also known in the artas Ruthenate(2−),bis[[2,2′-bipyridine]-4,4′-dimethanesulfonato(2−)-κN1,κN1′][1-[4-(4′-methyl[2,2′-bipyridin]-4-yl-κN1,κN1′)-1-oxobutoxy]-2,5-pyrrolidinedione]-,sodium (1:2), (OC-6-31), further known as Ruthenate(2−),bis[[2,2′-bipyridine]-4,4′-dimethanesulfonato(2−)-κN1,κN1′][1-[4-(4′-methyl[2,2′-bipyridin]-4-yl-κN1,κN1′)-1-oxobutoxy]-2,5-pyrrolidinedione]-,disodium, (OC-6-31)-(9CI));

BPRuUEEK-suberate-OSu (=CAS Reg. Number 406218-59-5 also known in theart as Ru(bpy)2-bpyCO-UEEK-suberate-OSu or Ruthenate(3−),bis(2,2′-bipyridine-κN1,κN1′[N-[4-(4′-methyl[2,2′-bipyridin]-4-yl-κN1,κN1′)-1-oxobutyl]-β-alanyl-L-α-glutamyl-L-α-glutamyl-N6-[8-[(2,5-dioxo-1-pyrrolidinyl)oxy]-1,8-dioxooctyl]-L-lysinato(3−)]-,(OC-6-33)-(9CI)), which is a BPRu-label with a peptide linker,U=beta-alanine, E=glutaminic acid, K=lysine;

BPRu-(UE)-25-K-suberate-OSu (=the suberate N-Hydroxysuccinimide esterderivative of CAS Reg. Number 406679-88-7, also known in the art asRuthenate(24−), bis(2,2′-bipyridine-κN1,κN1′)[N-[4-(4′-methyl[2,2′-bipyridin]-4-yl-κN1,κN1′)-1-oxobutyl]-(UE)₂₅-L-lysinato(26-)]-,(OC-6-33)-(9CI)), with U=β-alanyl, E=L-α-glutamyl;

BPRu2—SK2-suberate-OSu (=the suberate N-Hydroxysuccinimide esterderivative of CAS Reg. Number 406218-60-8, also known in the art asRuthenate(7−),bis(2,2′-bipyridine-κN1,κN1′)[N-[4-(4′-methyl[2,2′-bipyridin]-4-yl-κN1,κN1′)-1-oxobutyl]-β-alanyl-N6-(N-acetyl-(EU)₃)-L-lysyl-N6-(N-acetyl-(EU)₃)-L-lysyl-(UE)₂-L-lysinato(9-)]-,nonahydrogen (9CI)), with U=β-alanyl, E=L-α-glutamyl;4,4′,5′,5-tetramethyl bipyridine Re(I)(4-ethyl pyridine)(CO)₃ ⁺CF₃SO₃ ⁻,and Pt(2-(2-thienyl)pyridine)₂.

Further known chelates arebis[(4,4′-carbomethoxy)-2,2′-bipyridine]-2-[3-(4-methyl-2,2′-bipyridine-4-yl)propyl]-1,3-dioxolaneruthenium (II);bis(2,2′bipyridine)[4-(butan-1-al)-4′-methyl-2,2′-bipyridine] ruthenium(II); bis(2,2′-bipyridine)[4-(4′-methyl-2,2′-bipyridine-4′-yl)-butyricacid] ruthenium (II); (2,2′-bipyridine)[bis-bis(1,2-diphenylphosphino)ethylene]2-[3(4-methyl-2,2′-bipyridine-4′-yl)propyl]-1,3-di-oxolaneosmium (II);bis(2,2′-bipyridine)[4-(4′-methyl-2,2′-bipyridine)-butylamine]ruthenium(II);bis(2,2′-bipyridine)[1-bromo-4-(4′-methyl-2,2′-bipyridine-4-yl)-butane]ruthenium (II); and bis(2,2′-bipyridine) maleimido-hexanoic acid,4-methyl-2,2′-bipyridine-4′-butylamide ruthenium (II). In an embodiment,the ECL compound comprising a transition metal complex istris(2,2′-bipyridyl)ruthenium (Ru(bpy)₃ ²⁺, also known as Ru(bpy) orderivatives thereof like (BPRu═Ru(bpy)2-bpyCO—OSu), (Sulfo-BPRu NHSEster).

In a further embodiment, the ECL compound is selected from the groupconsisting of BPRu (═Ru(bpy)2-bpyCO—OSu); Sulfo-BPRu NHS Ester;BPRuUEEK-suberate-OSu; BPRu-(UE)-25-K-suberate-OSu andBPRu2—SK2-suberate-OSu.

It is known to a person skilled in the art that also hydrophilicderivatives of the aforesaid ECL compounds can be used. Therefore in anfurther embodiment the term ECL compound also includes hydrophilicderivatives of the aforesaid ECL compounds.

In a further embodiment, the ECL compound comprising a transition metalcomplex is an Ir-complex as disclosed in WO 2014/019707 (A2), in anembodiment is an Ir(6-phenylphenanthridine)₂-pyridine-2-carboxylic acidor a derivative thereof, including, e.g.Ir(6-phenylphenanthridine)₂-3-Hydroxypyridine-2-carboxylic acid,Ir(6-phenylphenanthridine)₂-4-(Hydroxymethyl)pyridine-2-carboxylic acid,Ir(6-phenylphenanthridine)2-2-(Carboxyethyl-phenyl)pyridine-2-carboxylicacid Ir(6-phenylphenanthridine)₂-5-(Methoxy)pyridine-2-carboxylic acid,or anIr(6-phenylphenanthridine)₂-2-(Carboxyethyl-phenyl)pyridine-2-carboxylicacid ester, or derivatives of it like iridium complexes with ligandssubstituted with one or more sulfonic acids or iridium complexes asdescribed in WO2012107419 (A1), WO2012107420 (A1), WO2014019707 (A2),WO2014019708 (A1), WO2014019709 (A2), WO2014019710 (A1), WO2014019711(A1).

In a further embodiment the ECL compound comprising a transition metalcomplex is CAS Registry Number 1556730-07-4 (=IB3/47, also known in theart as Iridate(3−),[5-[4-(2-carboxyethyl)phenyl]-2-pyridinecarboxylato(2−)-κN1,κO2]bis[2-(6-phenanthridinyl-κN)-5-(3-sulfonatopropoxy)phenyl-κC]—,cesium hydrogen (1:2:1) or the N-hydroxy succinimde ester thereof.

In an further embodiment the ECL compound comprising a transition metalcomplex is a Iridium complexes with two phenyl-phenanthridine ligandshaving two sulfonatopropoxy substituents, two sulfo-methyl, comprising2,9-Phenanthridinedimethanesulfonic acid, 6-phenyl-, sodium salt (CASRegistry Number 1554465-50-7) or two polyethylenglycol substituents, orthree of each, or combinations thereof.

In an further embodiment with Ruthenium complexes different linkers canbe used like (UE)25, or polyethylene glycol, or others.

In an embodiment, the compound comprising a transition metal complex isa detection reagent comprising an analyte specific reagent and a label.The term “analyte specific reagent” (ASR) according to the presentinvention has to be understood as a molecule or biomolecule (e.g. aprotein or antibody) with the capability to specifically bind theanalyte. “Analyte specific reagents” (ASRs) are a class of biologicalmolecules which can be used to identify and measure the amount of anindividual chemical substance in biological specimens. ASRs are forexample antibodies, both polyclonal and monoclonal, specific receptorproteins, ligands, nucleic acid sequences, and similar reagents which,through specific binding or chemical reaction with substances in aspecimen, are intended to use in a diagnostic application foridentification and quantification of an individual chemical substance orligand in biological specimens. An ASR will fulfill both, the criteriafor affinity as well as for specificity of binding the analyte. Certainanalytes are of high medical and diagnostic relevance even atconcentrations in the sub-picomolar range. Especially thegroup ofinfectious disease parameters such as hepatitis virus B soluble antigen(HBsAg), human immunodeficiency virus antigen (HIVAg), hepatitis C virusantigen (HCVAg), in particular hepatitis C virus core antigen (HCVcAg)and cardiac markers such as Troponin-T (TnT) are examples of suchanalytes. Especially in these cases, improving the sensitivity is ofmajor medical value for the patient.

The term “antibody” is used in the broadest sense and specificallyincludes monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), antibody fragments, single-chain antibodies,nanobodies, and the like. The term “antibody” encompasses the variousforms of antibody structures including whole antibodies and antibodyfragments. The antibody according to the invention is in one embodimenta human antibody, a humanized antibody, a chimeric antibody, an antibodyderived form other animal species like mouse, goat or sheep, amonoclonal or polyconal antibody, or a T cell antigen depleted antibody.Genetic engineering of antibodies is e.g. described in Morrison, S. L.,et al., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos.5,202,238 and 5,204,244; Riechmann, L., et al., Nature 332 (1988)323-327; Neuberger, M.S., et al., Nature 314 (1985) 268-270; Lonberg,N., Nat. Biotechnol. 23 (2005) 1117-1125.

Any antibody fragment retaining the above criteria of a analyte specificreagent can be used. Antibodies are generated by state of the artprocedures, e.g., as described in Tijssen (Tijssen, P., Practice andtheory of enzyme immunoassays, 11, Elsevier Science Publishers B. V.,Amsterdam, the whole book, especially pages 43-78). In addition, theskilled artisan is well aware of methods based on immunosorbents thatcan be used for the specific isolation of antibodies. By these means thequality of antibodies and hence their performance in immunoassays can beenhanced (Tijssen, P., supra, pages 108-115).

A “detection reagent” according to the present invention comprises ananalyte specific reagent (ASR) labeled with an electrochemiluminescentgroup, or an analyte homolog labeled with an electrochemiluminescentgroup. Depending on the test format, it is known to the skilled artisan,which type of detection reagent has to be selected for the various assayformats (e.g. sandwich assay, double antigen bridging assay (DAGS),competitive assay, homogeneous assay, heterogeneous assay). A detectionreagent in a heterogeneous immunoassay might be for example a labeledantibody. It is known to a person skilled in the art that the detectionreagent can be immobilized on a solid phase. In an embodiment the methodfor measuring an analyte in a sample via electrochemiluminescentdetection can be performed as a homogeneous assay. In an embodiment themethod can be performed as a heterogeneous assay. In an embodiment themethod can be performed in a sandwich assay format. In an embodiment themethod can be performed in a competitive assay format. Also in anembodiment the method can be performed in a double antigen bridgingassay format (DAGS). Known immunoassay formats are described in detailin the book of Price, C. P. and Newman, D. J., Principles and Practiceof Immunoassay, 2nd ed. (1997).

The term “branched-chain tertiary amine”, as used herein, relates to atertiary amine comprising at least one alkyl chain having a secondarycarbon atom in the alpha-position of the alkyl chain, i.e. to a tertiaryamine comprising at least one 1-branched alkyl chain. As used herein,the C-alpha atom of a side chain of a tertiary amine is the carbon atomcovalently bonded to the central nitrogen atom. Thus, in an embodiment,the branched-chain tertiary amine is an alpha-branched-chain tertiaryamine. In an embodiment, the branched-chain tertiary amine has thegeneral structure of formula (I)

wherein

at least one of R¹, R², and R³, in an embodiment one or two of R¹, R²,and R³ are independently selected side chains according to formula (II)

 wherein

-   -   m is 0, 1, or 2, in an embodiment is 0 or 1;    -   R⁴ is alkyl, in an embodiment is straight-chain C₁-C₃ alkyl, in        a further embodiment is ethyl or methyl, in another embodiment        is methyl,    -   R⁵ is alkyl, in an embodiment is straight-chain C1-C3 alkyl, in        another embodiment is ethyl or methyl, in an embodiment is        methyl,

and wherein the residual groups R¹, R², and R³ are independentlyselected from alkyl, in a further embodiment are independently selectedfrom straight-chain alkyl, in another embodiment are independentlyselected from the group consisting of n-pentyl, n-butyl, n-propyl, ethyland methyl, in an embodiment are independently selected from n-propyl,ethyl, and methyl.

As used herein, the terms “chemical compound”, “salt”, and “solvate” areused in their usual meaning known to the skilled chemist. If the netcharge of a compound according to the present invention is positive, inan embodiment counterions are trifluoromethanesulfonate (triflate),sulfate, alkyl sulfonate, tosylate, phosphate, tetrafluoroborate,hexafluorophosphate, trifluoracetate, perchlorate, chloride or nitrateions. If the net charge of a compound according to the present inventionis negative, in an embodiment counterions are lithium, sodium, and/orpotassium ions, or tetramethlyammonium ions. In an embodiment, the netcharge of a compound according to the present invention is the netcharge of the compound in aqueous solution under standard conditions asspecified elsewhere herein.

The term “side chain” is understood by the skilled person and relates toan atom or chemical group attached covalently to the core part of achemical compound as described herein, said core part also beingreferred to as “main chain” or “backbone”. In an embodiment, the sidechain is an organic side chain as described herein below. The term“substituted” side chain relates to a side chain substituted at one ormore positions, in an embodiment, at 1, 2, or 3 positions, whereinsubstituents may be attached at any available atom to produce a stablechemical compound. It is understood by the skilled person that the term“optionally substituted” side chain relates to an unsubstituted or to asubstituted side chain.

The term “organic side chain”, as used herein, relates to any,optionally substituted, side chain comprising at least one carbon atom.The term “alkyl”, as used herein, relates to a straight or branchedchain, saturated hydrocarbon group, linked to the main chain or to thecentral nitrogen of the tertiary amine by a covalent bond to at leastone of its at least one carbon atoms. Examples of alkyl groups arestraight chain alkyls, e.g., methyl, ethyl, n-propyl, n-butyl, n-pentyl,or branched chain alkyl groups, e.g., —CH(CH₃)₂, —CH(CH₂CH₃)₂,—CH(CH₃)(CH₂CH₃). Accordingly, alkyl groups include primary alkylgroups, secondary alkyl groups, and tertiary alkyl groups; in anembodiment, alkyl groups are primary alkyl groups or secondary alkylgroups.

In a further embodiment, at least one of the residual groups R¹, R², andR³ which are not selected according to formula (II) is straight-chainalkyl, in an embodiment is selected from the group consisting ofn-pentyl, n-butyl, n-propyl, ethyl or methyl, in further embodiment ispropyl, ethyl, or methyl. In a further embodiment, all of the residualgroups R¹, R², and R³ which are not selected according to formula (II)are straight-chain alkyl, in an embodiment are selected from the groupconsisting of n-pentyl, n-butyl, n-propyl, ethyl or methyl, in furtherembodiment is propyl, ethyl, or methyl.

In a further embodiment, the branched-chain tertiary amine is a compoundaccording to the general formula (III):

with

R² and R³ being selected independently from the group consisting ofmethyl, ethyl, n-propyl, and n-pentyl; and

R⁵ being selected from methyl, ethyl, and n-propyl. In a furtherembodiment, the branched-chain tertiary amine is a compound according tothe aforesaid general formula (III) with R² and R³ being selectedindependently from the group consisting of methyl, ethyl, n-propyl,n-butyl, and n-pentyl; and R⁵ being selected from methyl, ethyl, andn-propyl. Thus, in an embodiment, the branched-chain tertiary amine isone of the compounds of Table 1. In a further embodiment, thebranched-chain tertiary amine is one of the compounds of Table 1 or oneof compounds 46 to 60 of Table 2.

In a further embodiment, the branched-chain tertiary amine is a compoundaccording to the general formula (IV):

with

R³ being selected from the group consisting of methyl, ethyl, n-propyl,and n-pentyl, and

R⁵ and R⁶ being selected independently from methyl, ethyl, and n-propyl.In a further embodiment, the branched-chain tertiary amine is a compoundaccording to the aforesaid general formula (IV) with R³ being selectedfrom the group consisting of methyl, ethyl, n-propyl, n-butyl, andn-pentyl, and R⁵ and R⁶ being selected independently from methyl, ethyl,and n-propyl. Thus, in an embodiment, the branched-chain tertiary amineis one of the compounds of Table 2. In a further embodiment, thebranched-chain tertiary amine is one of compounds 31 to 45 or 61 to 75of Table 2.

In a further embodiment, the branched-chain tertiary amine is a compoundaccording to the general formula (V):

with R⁵, R⁶, and R⁷ being selected independently from methyl, ethyl andn-propyl. Thus, in an embodiment, the branched-chain tertiary amine isone of the compounds of Table 3.

In a further embodiment, the branched-chain tertiary amine is a compoundaccording to the general formula (VI):

with

R⁸ and R⁹ being independently selected from methyl, ethyl, n-propyl,n-butyl, n-pentyl, isopropyl, sec-butyl (1-methylpropyl), sec-pentyl(1-methylbutyl), and 3-pentyl (1-ethylpropyl). Thus, in an embodiment,the branched-chain tertiary amine is one of the compounds of Table 4.

In a further embodiment, the branched-chain tertiary amine is a compoundaccording to the general formula (VII):

wherein R¹⁰ to R¹⁵ are independently selected from —H and methyl,wherein at least one of R¹⁰ to R¹² is methyl; and wherein if R¹⁰, R¹¹and R¹² are methyl, at least one of R¹³ to R¹⁵ is methyl. Thus, in anembodiment, the branched-chain tertiary amine is compound No: 6, 7, 10,21, 22, 25, 32, 33, 36, 37, 38, 38, 42, 43,76, 78, or 81 of Tables 1 to4.

In an embodiment, the branched-chain tertiary amine isN,N-Dipropyl-N-(sec-butyl)amine (also known under its trivial name“Dipropyl-isobutylamine” or “DPIBA”, CAS 60021-91-2, compound No: 25 ofTable 1), N,N-Di(sec-butyl)-propylamine (also known under its trivialname “Diisobutyl-propylamine” or “DIBPA”, compound No: 43 of Table 2),or N,N-Diisopropyl-N-ethylamine (DIEA, CAS 7087-68-5, compound No: 32 ofTable 2). In a further embodiment, the branched-chain tertiary amine isN,N-Dipropyl-N-(sec-butyl)amine or N,N-Di(sec-butyl)-N-propylamine. In afurther embodiment, the branched-chain tertiary amine isN,N-Di(sec-butyl)-N-propylamine.

TABLE 1 embodiments of di(n-alkyl)-(1-methyl-alkyl)amines, substituentsrelate to formula (I) compound No R1 R2 R3 1 isopropyl methyl methyl 2isopropyl methyl ethyl 3 isopropyl methyl n-propyl 4 isopropyl methyln-butyl 5 isopropyl methyl n-pentyl 6 isopropyl ethyl ethyl 7 isopropylethyl n-propyl 8 isopropyl ethyl n-butyl 9 isopropyl ethyl n-pentyl 10isopropyl n-propyl n-propyl 11 isopropyl n-propyl n-butyl 12 isopropyln-propyl n-pentyl 13 isopropyl n-butyl n-butyl 14 isopropyl n-butyln-pentyl 15 isopropyl n-pentyl n-pentyl 16 sec-butyl methyl methyl(1-methylpropyl) 17 sec-butyl methyl ethyl (1-methylpropyl) 18 sec-butylmethyl n-propyl (1-methylpropyl) 19 sec-butyl methyl n-butyl(1-methylpropyl) 20 sec-butyl methyl n-pentyl (1-methylpropyl) 21sec-butyl ethyl ethyl (1-methylpropyl) 22 sec-butyl ethyl n-propyl(1-methylpropyl) 23 sec-butyl ethyl n-butyl (1-methylpropyl) 24sec-butyl ethyl n-pentyl (1-methylpropyl) 25 sec-butyl n-propyl n-propyl(1-methylpropyl) 26 sec-butyl n-propyl n-butyl (1-methylpropyl) 27sec-butyl n-propyl n-pentyl (1-methylpropyl) 28 sec-butyl n-butyln-butyl (1-methylpropyl) 29 sec-butyl n-butyl n-pentyl (1-methylpropyl)30 sec-butyl n-pentyl n-pentyl (1-methylpropyl)

TABLE 2 embodiments of (n-alkyl)-di(1-methyl-alkyl)amines and tertiarysec- pentyl-amines; substituents relate to formula (I) compound No R1 R2R3 31 isopropyl isopropyl methyl 32 isopropyl isopropyl ethyl 33isopropyl isopropyl n-propyl 34 isopropyl isopropyl n-butyl 35 isopropylisopropyl n-pentyl 36 sec-butyl isopropyl methyl (1-methylpropyl) 37sec-butyl isopropyl ethyl (1-methylpropyl) 38 sec-butyl isopropyln-propyl (1-methylpropyl) 39 sec-butyl isopropyl n-butyl(1-methylpropyl) 40 sec-butyl isopropyl n-pentyl (1-methylpropyl) 41sec-butyl sec-butyl methyl (1-methylpropyl) (1-methylpropyl) 42sec-butyl sec-butyl ethyl (1-methylpropyl) (1-methylpropyl) 43 sec-butylsec-butyl n-propyl (1-methylpropyl) (1-methylpropyl) 44 sec-butylsec-butyl n-butyl (1-methylpropyl) (1-methylpropyl) 45 sec-butylsec-butyl n-pentyl (1-methylpropyl) (1-methylpropyl) 46 sec-pentylmethyl methyl (1-methylbutyl) 47 sec-pentyl methyl ethyl (1-methylbutyl)48 sec-pentyl methyl n-propyl (1-methylbutyl) 49 sec-pentyl methyln-butyl (1-methylbutyl) 50 sec-pentyl methyl n-pentyl (1-methylbutyl) 51sec-pentyl ethyl ethyl (1-methylbutyl) 52 sec-pentyl ethyl n-propyl(1-methylbutyl) 53 sec-pentyl ethyl n-butyl (1-methylbutyl) 54sec-pentyl ethyl n-pentyl (1-methylbutyl) 55 sec-pentyl n-propyln-propyl (1-methylbutyl) 56 sec-pentyl n-propyl n-butyl (1-methylbutyl)57 sec-pentyl n-propyl n-pentyl (1-methylbutyl) 58 sec-pentyl n-butyln-butyl (1-methylbutyl) 59 sec-pentyl n-butyl n-pentyl (1-methylbutyl)60 sec-pentyl n-pentyl n-pentyl (1-methylbutyl) 61 sec-pentyl isopropylmethyl (1-methylbutyl) 62 sec-pentyl isopropyl ethyl (1-methylbutyl) 63sec-pentyl isopropyl n-propyl (1-methylbutyl) 64 sec-pentyl isopropyln-butyl (1-methylbutyl) 65 sec-pentyl isopropyl n-pentyl (1-methylbutyl)66 sec-pentyl sec-butyl methyl (1-methylbutyl) (1-methylpropyl) 67sec-pentyl sec-butyl ethyl (1-methylbutyl) (1-methylpropyl) 68sec-pentyl sec-butyl n-propyl (1-methylbutyl) (1-methylpropyl) 69sec-pentyl sec-butyl n-butyl (1-methylbutyl) (1-methylpropyl) 70sec-pentyl sec-butyl n-pentyl (1-methylbutyl) (1-methylpropyl) 71sec-pentyl sec-pentyl methyl (1-methylbutyl) (1-methylbutyl) 72sec-pentyl sec-pentyl ethyl (1-methylbutyl) (1-methylbutyl) 73sec-pentyl sec-pentyl n-propyl (1-methylbutyl) (1-methylbutyl) 74sec-pentyl sec-pentyl n-butyl (1-methylbutyl) (1-methylbutyl) 75sec-pentyl sec-pentyl n-pentyl (1-methylbutyl) (1-methylbutyl)

TABLE 3 embodiments of tri(1-methyl-alkyl)amines; substituents relate toformula (I) compound No R1 R2 R3 76 isopropyl isopropyl sec-butyl(1-methylpropyl) 77 isopropyl isopropyl sec-pentyl (1-methylbutyl) 78sec-butyl isopropyl sec-butyl (1-methylpropyl) (1-methylpropyl) 79sec-butyl isopropyl sec-pentyl (1-methylpropyl) (1-methylbutyl) 80sec-pentyl isopropyl sec-pentyl (1-methylbutyl) (1-methylbutyl) 81sec-butyl sec-butyl sec-butyl (1-methylpropyl) (1-methylpropyl)(1-methylpropyl) 82 sec-butyl sec-butyl sec-pentyl (1-methylpropyl)(1-methylpropyl) (1-methylbutyl) 83 sec-pentyl sec-pentyl sec-pentyl(1-methylbutyl) (1-methylbutyl) (1-methylbutyl)

TABLE 4 embodiments of tertiary (1-ethyl-alkyl)-amines: compound No R1R2 R3 84 3-pentyl methyl methyl (1-ethylpropyl) 85 3-pentyl methyl ethyl(1-ethylpropyl) 86 3-pentyl methyl n-propyl (1-ethylpropyl) 87 3-pentylmethyl n-butyl (1-ethylpropyl) 88 3-pentyl methyl n-pentyl(1-ethylpropyl) 89 3-pentyl ethyl ethyl (1-ethylpropyl) 90 3-pentylethyl n-propyl (1-ethylpropyl) 91 3-pentyl ethyl n-butyl (1-ethylpropyl)92 3-pentyl ethyl n-pentyl (1-ethylpropyl) 93 3-pentyl n-propyl n-propyl(1-ethylpropyl) 94 3-pentyl n-propyl n-butyl (1-ethylpropyl) 95 3-pentyln-propyl n-pentyl (1-ethylpropyl) 96 3-pentyl n-butyl n-butyl(1-ethylpropyl) 97 3-pentyl n-butyl n-pentyl (1-ethylpropyl) 98 3-pentyln-pentyl n-pentyl (1-ethylpropyl) 99 3-pentyl isopropyl methyl(1-ethylpropyl) 100 3-pentyl isopropyl ethyl (1-ethylpropyl) 1013-pentyl isopropyl n-propyl (1-ethylpropyl) 102 3-pentyl isopropyln-butyl (1-ethylpropyl) 103 3-pentyl isopropyl n-pentyl (1-ethylpropyl)104 3-pentyl sec-butyl methyl (1-ethylpropyl) (1-methylpropyl) 1053-pentyl sec-butyl ethyl (1-ethylpropyl) (1-methylpropyl) 106 3-pentylsec-butyl n-propyl (1-ethylpropyl) (1-methylpropyl) 107 3-pentylsec-butyl n-butyl (1-ethylpropyl) (1-methylpropyl) 108 3-pentylsec-butyl n-pentyl (1-ethylpropyl) (1-methylpropyl) 109 3-pentylsec-pentyl methyl (1-ethylpropyl) (1-methylbutyl) 110 3-pentylsec-pentyl ethyl (1-ethylpropyl) (1-methylbutyl) 111 3-pentyl sec-pentyln-propyl (1-ethylpropyl) (1-methylbutyl) 112 3-pentyl sec-pentyl n-butyl(1-ethylpropyl) (1-methylbutyl) 113 3-pentyl sec-pentyl n-pentyl(1-ethylpropyl) (1-methylbutyl) 114 3-pentyl isopropyl isopropyl(1-ethylpropyl) 115 3-pentyl isopropyl sec-butyl (1-ethylpropyl)(1-methylpropyl) 116 3-pentyl isopropyl sec-pentyl (1-ethylpropyl)(1-methylbutyl) 117 3-pentyl sec-butyl sec-butyl (1-ethylpropyl)(1-methylpropyl) (1-methylpropyl) 118 3-pentyl sec-butyl sec-pentyl(1-ethylpropyl) (1-methylpropyl) (1-methylbutyl) 119 3-pentyl sec-pentylsec-pentyl (1-ethylpropyl) (1-methylbutyl) (1-methylbutyl) 120 3-pentyl3-pentyl sec-butyl (1-ethylpropyl) (1-ethylpropyl) (1-methylpropyl) 1213-pentyl 3-pentyl sec-pentyl (1-ethylpropyl) (1-ethylpropyl)(1-methylbutyl) 122 3-pentyl 3-pentyl 3-pentyl (1-ethylpropyl)(1-ethylpropyl) (1-ethylpropyl)

In a further embodiment, the branched-chain amine of the presentinvention is an amine which, when oxidized by an effective amount ofelectrochemical energy, forms a strong reducing agent (“ElectrogeneratedChemiluminescence 69: The Tris(2,2′-bipyridine)ruthenium(II),(Ru(bpy)32+)/Tri-n-propylamine (TPrA) System Revisited”, Miao et al.(2002), Journal of the American Chemical Society 124(48):14478).

The term “composition” is known to the skilled person and relates to a,homogenous or inhomogenous, mixture of at least two chemical compounds.A “reagent composition” or “ECL-reagent composition” according to thepresent invention comprises reagents supporting ECL-signal generation,e.g. a coreactant, a buffering agent for pH control, and optionallyother components. The skilled artisan is aware of components of areagent composition which are required for ECL signal generation inelectrochemiluminescent detection methods. Also, as used herein, theterm “reaction mixture” relates to any mixture contacting a firstcompound with a second compound, e.g. a branched-chain tertiary amineand an ECL compound comprising a transition metal complex, allowing saidfirst and second compound to react. In an embodiment, the reactionmixture additionally comprises a solvent, in an embodiment compriseswater. In a further embodiment, the reaction mixture further comprisesone or more auxiliary compounds, e.g. a buffer, a preservative, or adetergent, or any combination thereof. In an embodiment, the reactionmixture further comprises compounds as described in WO 2012055815 A1. Inan embodiment, the reaction mixture is an aqueous solution. In anembodiment, a branched-chain tertiary amine according to the presentinvention is the only tertiary amine in the reaction mixture.

An “aqueous solution” as used herein is a homogeneous solution ofsubstances or liquid compounds dissolved in water. An aqueous solutionmay also comprise organic solvents. Organic solvents are known to theperson skilled in the art, e.g. methanol, ethanol or dimethylsulfoxid.As used herein it is also to be understood that an aqueous solution cancomprise at most 50% organic solvents. As will be understood by theskilled person, the term aqueous solution, in an embodiment, includes anaqueous solution into which particles are dispersed, in an embodimenthomogenously dispersed.

A species that participates with the ECL label in the ECL process isreferred to herein as ECL “coreactant”. Commonly used coreactants forECL include tertiary amines (e.g. tripropylamine (TPA)), oxalate, andpersulfate. The skilled artisan is aware of available coreactants usefulfor electrochemiluminescent detection methods. According to a specificembodiment the coreactant according to the present invention is abranched-chain tertiary amine.

The term “label” as used herein refers to any substance that is capableof producing a detectable electrochemiluminescent signal, whethervisibly detectable or detecable by using suitable instrumentation.Various labels suitable for use in the present invention include,electrochemiluminescent compounds. In an embodiment, the label is atransition metal complex or a detection reagent as specified above.

An “electrochemiluminescence assay” or “ECLA” is an electrochemicalassay in which an analyte molecule is detected by its binding to adetection reagent, which is linked to a label as specified above, and byinducing ECL to occur (“electrochemically triggering the release ofluminescence”). In an embodiment, an electrode electrochemicallyinitiates luminescence of the chemical label. In a further embodiment,ECL is triggered by applying a voltage to an electrode contacting areagent composition. For detecting an ECL signal, light emitted by theECL compound comprising a transition metal complex is measured by aphotodetector, indicating the presence or quantity of bound analytemolecule/target molecule complexes. ECLA methods are described, forexample, in U.S. Pat. Nos. 5,543,112; 5,935,779; and 6,316,607. Signalmodulation can be maximized for different analyte moleculeconcentrations for precise and sensitive measurements.

In an embodiment, electrochemically triggering the release ofluminescence comprises applying a measuring voltage at least 0.1 Vlower, in an embodiment at least 0.2 V lower, in a further embodiment atleast 0.3 V lower than the measuring voltage used in a comparableelectrochemical assay using tripropylamine as coreactant. The term“comparable electrochemical assay”, as used herein, relates to anelectrochemical assay comprising the use of at least the same ECLreagent and the same working electrode as the electrochemical assay ofthe present invention. In an embodiment, a comparable electrochemicalassay is an electrochemical assay comprising the same steps and reagentsas the electrochemical assay of the present invention with the exceptionof the coreactand, which is a branched-chain tertiary amine according tothe present invention, but TPA according to the comparableelectrochemical assay. Thus, in an embodiment, electrochemicallytriggering the release of luminescence comprises applying a measuringvoltage of from 0.7 V to 1.6 V, in an embodiment of from 0.8 V to 1.3 V,in a further embodiment of from 0.9 V to 1.1 V. According to the presentinvention, if not otherwise noted, potentials are measured versus astandard Ag/AgCl electrode, in an embodiment a Ag/AgCl electrode withsaturated KCl at 25° C. In an embodiment, the electrode contacting thereaction electrode of the present invention, which is also referred toas working electrode, comprises or consists of platinum (═Pt), gold(═Au), or glassy carbon, in an embodiment comprises or consists ofplatinum. Thus, in a further embodiment, triggering the release ofluminescence comprises applying a measuring voltage of from 0.8 V to 1.3V, in a further embodiment of from 0.9 V to 1.1 V versus an Ag/AgClelectrode at a platinum, gold, or glassy carbon working electrode, in anembodiment at a platinum working electrode. In a further embodiment, thebranched-chain amine is DIBPA and triggering the release of luminescencecomprises applying a measuring voltage of from 0.8 V to 1.2 V, in afurther embodiment of from 0.85 V to 1.1 V versus an Ag/AgCl electrodeat a platinum, gold, or glassy carbon working electrode, in anembodiment at a platinum working electrode. In a further embodiment, thebranched-chain amine is DPIBA and triggering the release of luminescencecomprises applying a measuring voltage of from 0.8 V to 1.2 V, in afurther embodiment of from 0.9 V to 1.1 V versus an Ag/AgCl electrode ata platinum, gold, or glassy carbon working electrode, in an embodimentat a platinum working electrode.

In an ECLA procedure, microparticles coated with detection reagent canbe suspended in the sample to efficiently bind the analyte and/or toallow efficient retrieval of bound analyte. For example, the particlescan have a diameter of 0.05 μm to 200 μm, 0.1 μm to 100 μm, or 0.5 μm to10 μm, and a surface component capable of binding an analyte molecule.In one frequently used ECLA-system (Elecsys®, Roche Dagnsotics,Germany), the microparticles have a diameter of about 3 μm. Themicroparticles can be formed of crosslinked starch, dextran, cellulose,protein, organic polymers, styrene copolymer such as styrene/butadienecopolymer, acrylonitrile/butadiene/styrene copolymer, vinylacetylacrylate copolymer, vinyl chloride/acrylate copolymer, inert inorganicparticles, chromium dioxide, oxides of iron, silica, silica mixtures,proteinaceous matter, or mixtures thereof, including but not limited tosepharose beads, latex beads, shell-core particles, and the like. Themicroparticles are, in an embodiment, monodisperse, and can be magnetic,such as paramagnetic beads. See, for example, U.S. Pat. Nos. 4,628,037;4,965,392; 4,695,393; 4,698,302; and 4,554,088. Microparticles can beused in an amount ranging from about 1 to 10,000 μg/ml, in an embodiment5 to 1,000 μg/ml.

A “sample”, as used according to the present invention, is obtained forthe purpose of an in vitro evaluation. As the skilled artisan willappreciate, any such assessment is made in vitro. If the sample is apatient sample, it is discarded afterwards. In an embodiment, thepatient sample is solely used for the in vitro diagnostic method of theinvention and the material of the patient sample is not transferred backinto the patient's body.

The embodiments of the invention can be used to test a sample for thepresence of an analyte or an activity of interest. Such samples may bein solid, emulsion, suspension, liquid, or gas form. They may be, butare not limited to, samples containing or derived from human or animals,for example, cells (live or dead) and cell-derived products,immortalized cells, cell fragments, cell fractions, cell lysates,organelles, cell membranes, hybridoma, cell culture supernatants(including supernatants from antibody producing organisms such ashybridomas), waste or drinking water, food, beverages, pharmaceuticalcompositions, blood, serum, plasma, hair, sweat, urine, feces, stool,saliva, tissue, biopsies, effluent, separated and/or fractionatedsamples, separated and/or fractionated liquids, organs, plants, plantparts, plant byproducts, soil, water, water supply, water sources,filtered residue from fluids (gas and liquid), swipes, absorbentmaterials, gels, cytoskeleton, unfractionated samples, unfractionatedcell lysates, cell nucleus/nuclei, nuclear fractions, chemicals,chemical solutions, structural biological components, skeletal(ligaments, tendons) components, separated and/or fractionated skeletalcomponents, hair fractions and/or separations, skin, skin samples, skinfractions, dermis, endodermis, eukaryotic cells, prokaryotic cells,fungus, yeast, immunological cells, drugs, therapeutic drugs, oils,extracts, mucous, sewage, environmental samples, organic solvents orair. In an embodiment the sample can further comprise, for example,water, alcohols, acetonitrile, dimethyl sulfoxide, dimethyl formamide,n-methyl-pyrrolidone, methanol or other organic solvents.

In an embodiment, the sample is a sample of a bacterium, anarchaebacterium, or a eukaryote. In an embodiment, the sample is asample of a mammal, in an embodiment, of a sheep, goat, cow, horse, pig,guinea pig, mouse, rat, cat dog, or human. In a further emboiment, thesample is a sample of a human. In an embodiment, the sample is a sampleof a body fluid, in an embodiment saliva, blood, plasma, serum, orurine; in an embodiment, the sample is cell-free. In a furtherembodiment, the sample is a tissue or organ sample, or an extractderived therefrom.

Analytes that may be measured include, but are not limited to, wholecells, cell surface antigens, protein complexes, cell signaling factorsand/or components, second messengers, second messenger signaling factorsand/or components, subcellular particles (e.g., organelles or membranefragments), viruses, prions, dust mites or fragments thereof, viroids,immunological factors, antibodies, antibody fragments, antigens,haptens, fatty acids, nucleic acids (and synthetic analogs), ribosomes,proteins (and synthetic analogs), lipoproteins, polysaccharides,inhibitors, cofactors, haptens, cell receptors, receptor ligands,lipopolysaccharides, glycoproteins, peptides, polypeptides, enzymes,enzyme substrates, enzyme products, nucleic acid processing enzymes(e.g., polymerases, nucleases, integrases, ligases, helicases,telomerases, etc.), protein processing enzymes (e.g., proteases,kinases, protein phophatases, ubiquitin-protein ligases, etc.), cellularmetabolites, endocrine factors, paracrine factors, autocrine factors,cytokines, hormones, pharmacological agents, drugs, therapeutic drugs,synthetic organic molecules, organometallic molecules, tranquilizers,barbiturates, alkaloids, steroids, vitamins, amino acids, sugars,lectins, recombinant or derived proteins, biotin, avidin, streptavidin,or inorganic molecules present in the sample.

Analytes which are whole cells may be animal, plant, or bacterial cells,and may be viable or dead. Examples include plant pathogens such asfungi and nematodes. The term “subcellular particles” is meant toencompass, for example, subcellular organelles, membrane particles asfrom disrupted cells, fragments of cell walls, ribosomes, multi-enzymecomplexes, and other particles which can be derived from livingorganisms. Nucleic acids include, for example, chromosomal DNA, plasmidDNA, viral DNA, and recombinant DNA derived from multiple sources.Nucleic acids also include RNAs, for example messenger RNAs, ribosomalRNAs and transfer RNAs. Polypeptides include, for example, enzymes,transport proteins, receptor proteins, and structural proteins such asviral coat proteins. In embodiments, polypeptides are enzymes andantibodies. In particular, polypeptides are monoclonal antibodies.Hormones include, for example, insulin and T4 thyroid hormone.Pharmacological agents include, for example, cardiac glycosides. It is,in an embodiment, within the scope of this invention to includesynthetic substances which chemically resemble biological materials,such as synthetic polypeptides, synthetic nucleic acids, and syntheticmembranes, vesicles and liposomes. The foregoing is not intended to be acomprehensive list of the biological substances suitable for use in thisinvention, but is meant only to illustrate the wide scope of theinvention.

Also, typically, the analyte of interest is present at a concentrationof 10⁻³ molar or less, for example, at least as low as 10⁻¹⁸ molar.

The expression “of interest” denotes an analyte or a substance ofpossible relevance that shall be analyzed or determined.

A “solid phase”, also known as “solid support”, is insoluble,functionalized, polymeric material to which detection reagents or otherreagents may be attached or covalently bound (often via a linker) to beimmobilized or allowing them to be readily separated (by filtration,centrifugation, washing etc.) from excess reagents, soluble reactionby-products, or solvents. Solid phases for the method according to theinvention are widely described in the state of the art (see, e.g.,Butler, J. E., Methods 22 (2000) 4-23). The term “solid phase” means anon-fluid substance, and includes particles (including microparticlesand beads) made from materials such as polymer, metal (paramagnetic,ferromagnetic particles), glass, and ceramic; gel substances such assilica, alumina, and polymer gels; capillaries, which may be made ofpolymer, metal, glass, and/or ceramic; zeolites and other poroussubstances; electrodes; microtiter plates; solid strips; and cuvettes,tubes, chips or other spectrometer sample containers. A solid phasecomponent of an assay is distinguished from inert solid surfaces withwhich the assay may be in contact in that a “solid phase” contains atleast one moiety on its surface, which is intended to interact with thedetection reagent. A solid phase may be a stationary component, such asa tube, strip, cuvette, chip or microtiter plate, or may be anon-stationary component, such as beads and microparticles.Microparticles can also be used as a solid phase for homogeneous assayformats. A variety of microparticles that allow either non-covalent orcovalent attachment of proteins and other substances may be used. Suchparticles include polymer particles such as polystyrene andpoly(methylmethacrylate); gold particles such as gold nanoparticles andgold colloids; and ceramic particles such as silica, glass, and metaloxide particles. See for example Martin, C. R., et al., AnalyticalChemistry-News & Features (1998) 322A-327A, which is incorporated hereinby reference.

The terms “chip”, “bio-chip”, “polymer-chip” or “protein-chip” are usedinterchangeably and refer to a collection of a large number of probes,markers or biochemical markers arranged on a shared substrate (e.g. asolid phase) which could be a portion of a silicon wafer, a nylon strip,a plastic strip, or a glass slide.

In an embodiment, the present invention relates to a method of detectingan electrochemiluminescence (ECL) signal comprising

a) contacting a reaction composition comprising

-   -   i) at least one branched-chain tertiary amine and    -   ii) an ECL compound comprising a transition metal complex

with an electrode,

b) electrochemically triggering the release of luminescence, and

c) detecting the ECL signal,

In a further embodiment, the present invention concerns a method fordetecting an analyte in a sample via electrochemiluminescence detection,comprising the steps of:

a) incubating the sample with a detection reagent labeled with anelectrochemiluminescent group comprising a transition metal complex, inan embodiment comprising a tris(2,2′-bipyridyl)ruthenium complex(Ru(bpy)₃ ²⁺),

b) separating analyte-bound and analyte-free labeled detection reagent,

c) contacting the separated analyte-bound labeled detection reagent witha branched-chain tertiary amine of the invention and with an electrode,

d) electrochemically triggering the release of luminescence, and

e) detecting the electrochemiluminescence (ECL) signal thereby detectingthe analyte.

In a further embodiment, the present invention relates to a method fordetecting an analyte in a sample via electrochemiluminescence detection,comprising the steps of:

a) incubating the sample with a detection reagent labeled with anelectrochemiluminescent group comprising a transition metal complex, inan embodiment comprising Ru(bpy)₃ ²⁺,

b) separating analyte-bound and analyte-free labeled detection reagent,

c) detecting ECL according to the method of the invention, with thedetection reagent being the compound comprising a transition metalcomplex, and

d) detecting the analyte based on the result of the ECL detection instep c).

An aspect of the invention relates to improved ECL methods based on thereagent compositions of the present invention, particularly ECL methodsfeaturing low detection limits. The reagent compositions comprising thebranched-chain tertiary amines of the present invention surprisinglyenable drastically reducing system background. More specifically, themethods of the invention provide improved sensitivity at low detectionlevels by reducing the background electrochemiluminescence in theabsence of ECL labels.

The inventors have surprisingly discovered that the use of certaincompounds from the group of branched-chain tertiary amine as coreactandsin ECL reactions provides a number of advantages, such as reducedoxidation potential of said compounds, and an improved signal/background(S/BG) ratio in ECL detection methods and thus improved ECL assayperformance.

A feature of the invention are methods for the determination of ananalyte in a sample to be investigated using an electrochemiluminescentlabel, wherein one of the following listed methods for measuringelectrochemiluminescent phenomena is employed.

Surprisingly the methods using compounds selected from the group ofbranched-chain tertiary amines emit less background luminescence thanconventional test reagents without these compounds. This is particularlyan advantage at low detection levels where increasing the signal tobackground ratio (=signal to noise ratio) greatly improves thesensitivity. Surprisingly, the inventors have found that performing anelectrochemiluminescent detection using a method according to thepresent invention results in an improved signal to noise ratio of ECLdetection.

The method for measuring an analyte in a sample viaelectrochemiluminescent detection according to the present invention canbe performed, in an embodiment, in an aqueous solution.

In an embodiment the branched-chain tertiary amine used in the method isselected from the group consisting of is N,N-Dipropyl-N-(sec-butyl)amine(DPIBA), N,N-Di(sec-butyl)-N-propylamine (DIBPA), orN,N-Diisopropyl-N-ethylamine (DIEA), in an embodiment, is DPIBA orDIBPA, in a further embodiment is DPIBA, in a further embodiment isDIBPA.

For the avoidance of doubt, in an embodiment, the branched-chaintertiary amine is not tripropyl amine, triethanol amine, triethyl amine1,4-piperazine-bis(ethane-sulfonic acid), 1-piperidine ethanol,1,4-diazabicyclo(2.2.2)octane, triisopropyl amine, or dibutylethanolamine.

In an embodiment, the branched-chain tertiary amine is used in themethod in a concentration of at least 50 mM (i.e. 50 mmol/l), in afurther embodiment in a concentration of at most 500 mM, in a furtherembodiment in a concentration of 50 mM to 500 mM, in a furtherembodiment in a concentration of 75 mM to 350 mM, in a furtherembodiment in a concentration of 100 mM to 250 mM.

In an embodiment the method according to the present invention isparticularly well suited to detect biomolecules, such as proteins,polypeptides, peptides, peptidic fragments, hormones, petid hormones,vitamins, provitamins, vitamin metabolites and amino acids in a sampleof interest. In a further embodiment, the method according to thepresent invention is particularly well suited to detect biomoleculesfrom the further classes of steroids, drugs, and therapeutics.

The sample used in the methods according to the present invention is inan embodiment a liquid sample, e.g., whole blood, serum or plasma. Thesample, or more specific the sample of interest, in an embodiment maycomprise any body fluid and stool. In an embodiment the sample will be aliquid sample like saliva, stool extracts, urine, whole blood, plasma orserum. In an embodiment the sample will be whole blood, plasma or serum.

It is known to a person skilled in the art that, in an embodiment, allsteps in the methods of the present invention can be performed in thesame location, e.g. in the same reaction vessel. Said steps may also beperformed in an automatic process controlled by a control means.

Unspecific sample components and analyte-free labeled detection reagentcan be removed in a separation process. For example, analyte-bound andanalyte-free labeled detection reagent can be separated using a washingstep. The analyte-bound labeled detection reagent is then incubated toperform the ECL detection of the method.

Also other test components supporting the electrochemiluminscentdetection of an analyte may be used in the methods according to thepresent invention.

It has been found by the inventors that the background (BG) generated bytertiary amines is an exponential function of the voltage applied fortheir oxidation. Furthermore the inventors found that branched-chaintertiary amines exhibit an oxidation potential significantly lower ascompared to unbranched analogues, in particular tripropylamine (TPA).Accordingly the inventors found, that a method for measuring an analytein a sample via electrochemiluminescent detection using branched-chaintertiary amines requires a reduced oxidation potential, improving signalto noise ratio (S/BG ratio) in ECL detection. In an embodiment, the S/BGratio is improved by at least a factor of 1.2, in a further embodimentby at least a factor of 1.4. Moreover, it was found that the improvingeffect can be further enhanced by using Iridium labels. The accumulatedeffect of branched-chain tertiary amine and Iridium labels in a reagentcomposition leads to at least 1.3-fold improved S/BG ratio in ECLdetection. Moreover, since the ECL reaction according to the presentinvention can be performed at a lower voltage, wearing in the workingelectrode is also reduced.

In an embodiment the present invention concerns a method for measuringan analyte in a sample via electrochemiluminescent detection, comprisingthe steps of a) incubating the sample with a detection reagent labeledwith an electrochemiluminescent group, b) separating analyte-bound andanalyte-free labeled detection reagent, c) incubating the separatedlabeled detection reagent with a reagent composition comprising i) atleast one branched-chain tertiary amine as coreactant, and ii) Ru(bpy)₃²⁺, d) electrochemically triggering the release of luminescence, and e)determining the electrochemiluminescence (ECL) signal thereby measuringthe analyte.

In an embodiment, the methods of the present invention are characterizedin that the reagent composition comprises in addition a detergent and/ora buffering agent.

In an embodiment, the methods of the present invention are characterizedin that the reagent composition comprises in addition a salt and/or ananti-foam agent.

In an embodiment, the invention relates to a method for conducting anelectrochemiluminescence assay wherein electrochemiluminescence isinduced in the presence of a reagent composition according to thepresent invention.

A typical ECL measurement process for an ECL immunoassay comprisesmultiple exchanges of liquids and/or mixtures in the ECL measurementcell (e.g. a flow cell). In an embodiment, a typical ECL measurementprocess consists of several steps explained below.

The skilled artisan is aware that an ECL measurement cell, in anembodiment, is conditioned or regenerated before the ECL detection steptakes place by rinsing said ECL measurement cell with a reagentcomposition according to the present invention and additional theapplication of an electric potential. This step, in an embodiment, isone part of the process of determining analytes using ECL. It has beendescribed in EP 1 051 621 that during this conditioning step a layer isformed on the surface of the measurement electrode(s) supporting thesignal generation during the measurement of an analyte in an ECLmeasurement cell.

In an embodiment of an ECL measurement process, a reagent mixture isintroduced into the cleaned and conditioned ECL measurement cell throughthe fluid inlet channel into the ECL measurement cell cavity, whereinsaid mixture comprises constituents of the sample, reagents and magneticparticles. Said mixture introduced into the measurement cell may besurrounded by a reagent composition according to the present inventionflowing in front and after said mixture.

In an embodiment, in such an ECL immunoassay a detection reagentcomprising complex-molecules which are labeled with anelectrochemiluminescent group and which are characteristic for theanalysis, are bound to these magnetic particles by a pair of specificbiochemical binding partners, e.g. streptavidin and biotin. The magneticparticles are for example coated with streptavidin-polymer, whereasbiotin is bound to the complex-molecules.

In an embodiment, in the ECL measurement cell the magnetic particles aretrapped to the surface of an electrode together with the labeledcomplex-molecules bound thereto in the magnetic field of a magnetarranged close to said electrode. The magnetic field is applied during acontinuous flow of the mixture, whereby incubate and/or reagentcomposition discharges from the ECL measurement cell cavity through thefluid outlet channel.

After trapping the magnetic particles, a reagent composition accordingto the present invention containing an ECL coreactant is introduced intothe ECL measurement cell in a next step, whereby the magnetic particlesare washed by said reagent composition. This step of washing is toremove unbound components of said incubate from the electrode whichpotentially interfere with the electrochemical reaction. In anembodiment, the washing step may also be performed before contacting themagnetic particles with a reagent composition according to the presentinvention, e.g. with a washing buffer (pre-wash method).

Thereafter the release of the electrochemiluminescence (ECL) signal iselectrochemically triggered by application of an electric potential,whereby the intensity of the luminescence light is detected by means ofa photosensor and may be evaluated as a measure for the concentration ofthe labeled complex-molecules on the magnetic particles located at thesurface of the electrode, whereby this concentration again serves as ameasure for the concentration of the analyte in the sample.

After the electrochemiluminescence detection the ECL measurement cellusually is rinsed with a cleaning fluid.

An apparatus for carrying out detection methods by means ofelectrochemiluminescence is mentioned in the example section (Examples2, 3, and 4) or described in EP 1 892 524 (A1). Moreover, such anapparatus can comprise means for controlling the temperature of themeasuring unit and/or a liquid vessel. The measuring unit is understoodto be a cell in which the electrochemiluminescence is measured. Theliquid vessel can be a storage container, but also a feeding device; forexample, a tube for the reagent solution, contained in the measuringunit during the measurement.

An aspect of the invention relates to improved reagent compositions forECL-signal generation, in particular those which lead to enhanced signalto noise ratios (S/BG ratio). More specifically, the reagentcompositions of the invention provide improved sensitivity at lowdetection levels by reducing the background electrochemiluminescence inthe absence of ECL labels. Surprisingly a reagent composition comprisingbranched-chain tertiary amines emit less background luminescence thanconventional test reagents without these compounds. This is particularlyan advantage at low detection levels where increasing the signal tobackground ratio (=signal to noise ratio) greatly improves thesensitivity. This improved reagent composition contains a compound fromthe group of branched-chain tertiary amine as well as optional furtherECL supporting reagents. Surprisingly the inventors have found thatperforming an electrochemiluminescent detection using a reagentcomposition according to the present invention results in a improvedsignal to noise ratio (S/BG ratio) of ECL detection as specified hereinabove.

An aspect of the invention relates to a reagent composition that giveshigh signal to background ratios in electrochemiluminescence assays. Thesignal difference between specific signals and background signals isincreased. Such improved properties have been achieved through theidentification of advantageous combinations of ECL coreactant, pHbuffering agents, detergent and pH and, in particular, through the useof branched-chain tertiary amines as coreactands.

The reagent composition provides a suitable environment for efficientlyinducing ECL labels to emit ECL and for sensitively measuring ECL labelsvia the measurement of ECL. The reagent composition of the invention mayoptionally comprise additional components including preservatives,detergents, anti-foaming agents, ECL active species, salts, acidic andbasic compounds for pH control (buffering agents), metal ions and/ormetal chelating agents. The reagent composition of the invention mayalso include components of a biological assay, which in some cases maybe labeled with an ECL label, including binding reagents, enzymes,enzyme substrates, cofactors and/or enzyme inhibitors. The inventionalso includes assay reagents, compositions, kits, systems and systemcomponents that comprise the reagent composition of the invention and,optionally, additional assay components. The invention also includesmethods for conducting ECL assays using the reagent composition of theinvention.

In an embodiment the current invention relates to a reagent compositionfor detecting ECL, comprising

i) a branched-chain tertiary amine as coreactant, and

ii) optionally a further ECL reagent.

A definition and embodiments of the branched-chain tertiary amine areprovided elsewhere herein. In an embodiment, the branched-chain tertiaryamine of the reagent composition is selected from the group consistingof N,N-Dipropyl-N-(sec-butyl)amine (DPIBA, compound No: 25 of Table 1),N,N-Di(sec-butyl)-propylamine (DIBPA, compound No: 43 of Table 2), andN,N-Diisopropyl-ethylamine (DIEA, CAS 7087-68-5, compound No: 32 ofTable 2). In a further embodiment, the branched-chain tertiary amine isN,N-Dipropyl-N-(sec-butyl)amine or N,N-Di(sec-butyl)-propylamine. In afurther embodiment, the branched-chain tertiary amine isN,N-Di(sec-butyl)-propylamine.

In an embodiment, the concentration of the branched-chain tertiary amineis selected optimally for the ECL enhancing effect, e.g. as shown in theExamples. Methods to determine the optimal concentration for abranched-chain tertiary amine in the reagent composition or reactioncomposition are known to the skilled artisan.

In an embodiment, the reagent composition comprises the branched-chaintertiary amine in a concentration of 50 mM to 500 mM. In a furtherembodiment the reagent composition comprises the branched-chain tertiaryamine in a concentration of 75 mM to 350 mM. In a further embodiment,the reagent composition comprises the branched-chain tertiary amine in aconcentration of 100 mM to 250 mM. It will be realized by the skilledperson that, in case the reagent composition is provided as a stocksolution, said stock solution may require dilution in order to obtain areaction composition and that the concentration of constituents of sucha stock solution may be adjusted according to the dilution factorintended. Thus, the aforesaid concentrations, in an embodiment, arefinal concentrations of the branched-chain tertiary amine in a reactioncomposition.

In an embodiment, the reagent composition of the present inventioncomprises a further ECL reagent. As used herein, the term “ECL reagent”includes ECL supporting reagents as well as ECL compounds comprising atransition metal complex, as specified elsewhere herein. ECL supportingreagents are, in principle, known to the skilled person. In anembodiment, an ECL supporting reagent is a preservative, a buffercompound, a detergent, an inorganic salt, in particular a sodiumhalogenide, or an anti-foaming agent, or any combination thereof. In anembodiment, the ECL supporting reagent is or comprises a carboxamide oramide. The term “carboxamide”, in an embodiment, relates to acarboxamide described in WO 2012/055815 A1, in an embodiment relates to,optionally halogenated, acetamide, propanamide, or butyramide, in afurther embodiment is propanamide.

It may be beneficial when storing a reagent composition to include apreservative that prevents microbial growth. Additionally, suitablepreservatives are identified to control bacterial and fungal growth toenable long term storage and use of the reagent composition. The reagentcomposition according to the present invention may additionally containone or more preservatives. In an embodiment of the present invention thereagent composition comprises a preservative (preservative agent) asspecified elsewehere herein.

In an embodiment, the reagent composition further comprises a detergent.Suitable detergents for a reagent composition according to the presentinvention are those from the group consisting of fatty acid alcoholethoxylates, including poly(ethylene glycol)ethers, for examplepolidocanol or other poly(ethylene glycol)ethers with the formulaC_(X)EO_(Y) with X=8-18 and Y=2-9, genapol (isotridecylpoly((ethyleneglycol ether)_(n)), Plantaren® (alkylpolygluco side), octylgluco side(octyl-beta-D-glucopyranoside) as well as zwitterionic detergents likeZwittergent 3-12 or a mixture thereof. The detergents are used inconcentrations ranging between 0.01% and 2%. The optimal concentrationcan be easily determined for each detergent. The most suitableconcentrations are those ranging between 0.05% and 1%. In an embodimentthe reagent composition according to the present invention comprisesdetergents selected from the group consisting of polidocanol or otherpoly(ethylene glycol)ethers with the formula C_(X)EO_(Y) with X=8-18 andY=2-9, octylglucoside (octyl-beta-D-glucopyrano side) or zwitterionicdetergents like Zwittergent 3-12 or a mixture thereof. In an embodimentthe reagent composition comprises detergents selected from the groupconsisting of polydocanol, octylgluco side (octyl-beta-D-glucopyranoside) and Zwittergent 3-12, or a mixture thereof.

Further, in an embodiment the electrochemiluminescent signal can also beincreased by adjusting the pH to a value between 6.0 and 8.0, in anembodiment between 6.0 and 7.5, in an embodiment between 6.2 and 6.9.This can be done conventionally by using a pH buffering agent suitablefor this range, known to a person skilled in the art. In an embodimentthe buffering agent suitable for the reagent composition comprises KOHand phosphoric acid (H₃PO₄); in an embodiment, the buffer is a sodium orpotassium phosphate buffer, in an embodiment, a potassion phosphatebuffer.

Furthermore, the signal can be increased by adding salts, includinginorganic salts like, for example NaBr, NaCl, NaJ. The salts, especiallyNaCl, are added in concentrations ranging between 1 mM and 1 M, in anembodiment between 10 mM and 100 mM, in a further embodiment between 10mM and 50 mM.

It may be beneficial, especially in HTS applications, to avoid theproduction of bubbles or foam. For this reason it may be desirable toadd anti-foaming agents to a reagent composition. Many commercialantifoaming agents (including Antifoams o-30, Antifoam 204, Antifoam A,Antifoam SE-15, Antifoam SO-25 and Antifoam 289) may be added to thereagent composition according to the present invention.

The reagent composition of the invention may further include an ECLcompound comprising a transition metal complex as specified elsewhereherein. The ECL compound comprising a transition metal complex may be aconventional ECL label. Examples of ECL labels includetris-bipyridyl-ruthenium (RuBpy) and other organometallic compoundswhere the metal is from, for example, the metals of group VII and VIII,including Re, Ru, Jr and Os. These ECL labels are used by a personskilled in the art to label an analyte specific reagent with anelectrochemiluminescent group, or to label the analyte itself with anelectrochemiluminescent group. In an embodiment the reagent compositionof the invention contains an analyte labeled with an ECL compoundcomprising a transition metal complex and/or an analyte specific reagentlabeled with an ECL compound comprising a transition metal complex asspecified elsewhere herein.

The reagents and mixtures thereof used in the reagent composition may beprovided either in liquid, frozen, deep frozen, vaporize frozen,lyophilized, gas, solid or dried form before usage. Before usage of thereagent composition, the reagents are, in an embodiment, dissolved in asolvent, in an embodiment in water.

The reagent compositions of the present invention are of particularvalue in high sensitivity assays. In some embodiments of the invention,the performance of ECL assays is improved even further through optimalcombinations of reagent composition with electrode composition. In anembodiment, ECL electrodes for use in conjunction with the means andmethods of the present invention comprise or consist of Au, Ir, Pt orCarbon, in an embodiment including boron-doped diamond electrodes.

For the determination of ECL, the reagent composition according to thepresent invention may be mixed with additional compounds forming areaction composition. In an embodiment, the current invention relates toa reaction composition for determining ECL, comprising i) at least oneECL compound comprising a transition metal complex ii) at least onebranched-chain tertiary amine as a coreactant. In an embodiment, thereaction composition further comprises an analyte.

In an embodiment, the present invention relates to an ECL reactioncomposition comprising i) at least one ECL compound comprising atransition metal complex, ii) at least one branched-chain tertiary amineas a coreactant, iii) an analyte, and iv) at least one analyte-specificreagent.

As will be appreciated by the skilled person, the at least one ECLcompound comprising a transition metal complex and the at least oneanalyte-specific reagent may be covalently linked, i.e. may together bea detection reagent. It is, however, also envisaged that the ECLcompound comprising a transition metal complex is bound to an agentspecifically binding to the analyte-specific reagent. As will beappreciated by the skilled person, in an embodiment, the aforesaidreaction composition is a composition after removal of non-specificallybound ECL compound comprising a transition metal complex from thereaction mixture.

In a further embodiment, the reaction composition is a reactioncomposition for determining ECL, comprising i) at least one ECL compoundcomprising a transition metal complex covalently coupled to an analyteor to a structural analog of the analyte, ii) at least onebranched-chain tertiary amine as a coreactant, and iii) an analyte.

In an embodiment, the aforesaid reaction compositions further compriseat least one further ECL supporting reagent as specified above.

An aspect of the present invention relates to the use of abranched-chain tertiary amine, of a reagent composition of theinvention, or of a reaction composition of the invention, for performingan electrochemiluminescent detection method.

One aspect of the invention relates to kits comprising, in one or morecontainers, a branched-chain tertiary amine and an ECL reagent asspecified herein above. The components may be optionally combined withadditional reagents, to form the reagent composition or the reactioncomposition of the invention. The kits may also comprise in anembodiment additional assay related components such as a diluent, awashing solution, a protein denaturating reagent, one or more enzymes, abinding reagent, an assay plate, disposables, electrodes etc.

The term “kit”, as used herein, refers to a collection of theaforementioned compounds, means or reagents of the present inventionwhich may or may not be packaged together. The components of the kit maybe comprised by separate vials (i.e. as a kit of separate parts) orprovided in a single vial. Moreover, it is to be understood that the kitof the present invention is, in an embodiment, to be used for practicingthe methods referred to herein above. It is, in an embodiment, envisagedthat all components are provided in a ready-to-use manner for practicingthe methods referred to above. Further, the kit, in an embodiment,contains instructions for carrying out said methods. The instructionscan be provided by a user's manual in paper- or electronic form. Inaddition, the manual may comprise instructions for interpreting theresults obtained when carrying out the aforementioned methods using thekit of the present invention.

In an embodiment, the chemical agents of the kit are contained in one ormore glass or plastic containers, appropriately labeled with informationregarding the contents and instructions regarding proper storage anduse. Further information, which may relate to contents, lot number,production date, best before date, instructions regarding proper storageand use may be also stored on a RFID chip placed on the glass oderplastic containers. The information stored on such RFID chip can be readby an antenna connected to a RFID reader device and further processed ina control means.

In an embodiment some or all of the components of the reagentcomposition may be stored in an embodiment in a liquid or dry state.

In an embodiment the present invention concerns a kit for measuring ECL,which comprises a reagent composition for determining ECL as specifiedherein above.

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theembodiments and claims. It is understood that modifications can be madein the procedures set forth without departing from the spirit of theinvention.

A further aspect of the invention relates to an ECL device comprising abranched-chain tertiary amine as specified herein above.

The term “device”, as used herein, relates to a system of means adaptedfor detecting ECL, further comprising at least the aforementioned meansoperatively linked to said device as to allow the result of thedetection to be obtained. Typical means for operatively linking abranched-chain tertiary amine to the device of the present invention arecontainers linked to pumps, which, in an embodiment, mediate transfer ofsaid branched-chain tertiary amine into a measuring chamber. How to linkthe means in an operating manner will depend on the type of meansincluded into the device. In an embodiment, the means are comprised by asingle device.

In an embodiment, the device comprises a sample treatment unitcomprising a receptacle for a sample. The receptacle may directlycontact the sample, or may be a receptacle for a further means receivingthe sample, wherein the further means may be e.g. a multi-well plate, towhich a sample or a multiplicity of samples may be applied. Moreover,the sample treatment unit, in an embodiment, comprises a branched-chaintertiary amine, e.g. in a dry form or in a reservoir connected to adosing means, e.g. a tubing connected to a pump. In an embodiment, thesample treatment unit comprises at least one ECL compound comprising atransition metal complex, e.g. in a dried form or in a reservoirconnected to a dosing means, e.g. a tubing connected to a pump. In afurther embodiment, the sample treatment unit comprises means for mixingand means for adjusting the temperature of a reaction mixture.Optionally, the sample treatment unit may comprise a washing means forremoving unspecifically bound ECL compound comprising a transition metalcomplex and/or binding agent.

In an embodiment, the result of the detection may be obtained in ananalyzing unit of the device by visual inspection by the user or byperforming a detection measurement on an appropriate device. In anembodiment, the analyzing unit of the device of the present inventionfurther comprises a detection unit for detecting ECL according to thepresent invention. Means suitable as a detection unit according to thepresent invention are known to the skilled person and include, e.g.photometric devices, in particular luminometric devices.

In an embodiment, the device of the present invention further comprisesa data output unit, connected to the detection unit. The data outputunit, in an embodiment, is adapted to output data obtained by thedetection unit. Suitable data output units are known to the skilledperson and include simple output units such as an indicator lamp or adisplay indicating that ECL was detected above the detection threshold.An output unit may, however, also be an interface to an evaluationdevice, wherein said interface may be any kind of means of transferringdata, including, e.g. cable connections like USB, wireless connectionslike wireless LAN, bluetooth, and the like, or indirect connections suchas data transfer by instant messaging, email, or the like.

In an embodiment, the device of the present invention is part of ananalytic system, said analytic system further comprising an evaluationdevice. As will be understood by the skilled person, the evaluationdevice may be comprised in the same housing as the device of theinvention, e.g. as an evaluation unit, or may be a separate device. Inan embodiment, the evaluation device comprises a microprocessorprogrammed to receive output data from an output unit of the device ofthe present invention and to perform logical operations providing anevaluation of said output data. Evaluation of output data may comprise,e.g., correcting data for values measured in one or more controldetection reaction, statistical calculations, e.g calculating means oftwo or more parallel detection reactions, correcting data for dilutionfactors, comparing output data to reference values, compiling data in alist, and the like. In an embodiment, the evaluation device furthercomprises a data storage unit. In a further embodiment, said datastorage unit comprises reference values, e.g. in a reference value database. Moreover, in an embodiment, the data storage unit is adapted tostore output data received from a device of the present invention, asspecified above.

In an embodiment, where means for automatically detecting ECL areapplied, the data obtained by said automatically operating means can beprocessed by, e.g., a computer program in order to establish adiagnosis. Typical means for detection are disclosed in connection withembodiments relating to the methods of the invention above. In such acase, the means are operatively linked in that the user of the systembrings together the result of the determination of ECL and thediagnostic value thereof due to the instructions and interpretationsgiven in a manual. The person skilled in the art will realize how tolink the means without further inventive skills. Typical devices arethose which can be applied without the particular knowledge of aspecialized clinician, e.g., test stripes or electronic devices whichmerely require loading with a sample. The results may be given as outputof parametric diagnostic raw data, in an embodiment, as absolute orrelative amounts. It is to be understood that these data will needinterpretation by the clinician. However, also envisaged are expertsystem devices wherein the output comprises processed diagnostic rawdata the interpretation of which does not require a specializedclinician.

In view of the above, the following embodiments are particularlyenvisaged:

1. A method of detecting an electrochemiluminescence (ECL) signalcomprising

a) contacting a reaction composition comprising

-   -   i) at least one branched-chain tertiary amine and    -   ii) an ECL compound comprising a transition metal complex    -   with an electrode,

b) electrochemically triggering the release of luminescence, and

c) detecting the ECL signal.

2. The method of embodiment 1, wherein said branched-chain tertiaryamine has the general structure of formula (I)

wherein

at least one of R¹, R², and R³, in an embodiment one or two of R¹, R²,and R³ are independently selected side chains according to formula (II)

 wherein

-   -   m is 0, 1, or 2, in an embodiment is 0 or 1;    -   R⁴ is alkyl, in an embodiment is straight-chain C₁-C₃ alkyl, in        a further embodiment is ethyl or methyl, in another embodiment        is methyl,    -   R⁵ is alkyl, in an embodiment is straight-chain C1-C3 alkyl, in        another embodiment is ethyl or methyl, in an embodiment is        methyl,

and wherein the residual groups R¹, R², and R³ are independentlyselected from alkyl, in a further embodiment are independently selectedfrom straight-chain alkyl, in another embodiment are independentlyselected from the group consisting of n-pentyl, n-butyl, n-propyl, ethyland methyl, in an embodiment are independently selected from n-propyl,ethyl, and methyl.

3. The method of embodiment 1 or 2, wherein at least one of the residualgroups R¹, R², and R³ not being selected according to formula (II) isstraight-chain alkyl, in an embodiment is selected from the groupconsisting of n-pentyl, n-butyl, n-propyl, ethyl or methyl, in a furtherembodiment is propyl, ethyl, or methyl.

4. The method of any one of embodiments 1 to 3, wherein saidbranched-chain tertiary amine is one of compounds 1 to 122 of any one ofTables 1 to 4.

5. The method of any one of embodiments 1 to 4, wherein saidbranched-chain tertiary amine is one of compounds 1 to 83 of any one ofTables 1 to 3.

6. The method of any one of embodiments 1 to 5, wherein saidbranched-chain tertiary amine is one of compounds 1 to 75 of Table 1 or2.

7. The method of any one of embodiments 1 to 6, wherein saidbranched-chain tertiary amine is N,N-Dipropyl-N-(sec-butyl)amine(DPIBA), N,N-Di(sec-butyl)-N-propylamine (DIBPA), orN,N-Diisopropyl-N-ethylamine (DIEA), in an embodiment, is DPIBA orDIBPA, in a further embodiment is DPIBA, in a further embodiment isDIBPA.

8. The method of any one of embodiments 1 to 7, wherein saidbranched-chain tertiary amine is not triisopropylamine.

9. The method of any one of embodiments 1 to 8, wherein said electrodecomprises or consists of Au, Ir, Pt or Carbon, in an embodiment is aboron-doped diamond electrode or a glassy carbon electrode; in anembodiment comprises or consist of platinum, gold, or glassy carbon, inan embodiment comprises or consists of platinum.

10. The method of any one of embodiments 1 to 9, wherein saidelectrochemically triggering the release of luminescence comprisesapplying a potential at the working electrode of from 0.8 V to 1.3 V, inan embodiment of from 0.9 V to 1.1 V versus an Ag/AgCl-electrode.

11. The method of any one of embodiments 1 to 10, wherein saidtransition metal complex comprises at least one of Ruthenium ions,Iridium ions, Rhenium ions, Osmium ions, Europium ions, Terbium ions,and Dysprosium ions; in an embodiment, wherein said transition metalcomplex comprises Ruthenium ions or comprises Iridium ions, in a furtherembodiment comprises Iridium ions.

12. The method of any one of embodiments 1 to 11, wherein said compoundcomprising a transition metal complex is selected from the listconsisting of Ru(bpy)₃ ²⁺, Ru(bpy)2-bpyCO—OSu), Sulfo-BPRu NHS Ester,BPRuUEEK-suberate-OSu, BPRu-(UE)-25-K-suberate-OSu,BPRu2—SK2-suberate-OSu, 4,4′,5′,5-tetramethyl bipyridine Re(I)(4-ethylpyridine)(CO)₃ ⁺CF₃SO₃ ⁻, Pt(2-(2-thienyl)pyridine)2,Ir(6-phenylphenanthridine)₂-2-(Carboxyethyl-phenyl)pyridine-2-carboxylicacid ester, and hydrophilic derivatives thereof.

13. The method of any one of embodiments 1 to 12, wherein said compoundcomprising a transition metal complex is selected from the listconsisting of Ru(bpy)₃ ²⁺, Ru(bpy)₂-bpyCO—OSu, Sulfo-BPRu NHS Ester,Ir(6-phenylphenanthridine)₂-2-(Carboxyethyl-phenyl)pyridine-2-carboxylicacid ester, or a hydrophilic derivative thereof, in an embodiment isIr(6-phenylphenanthridine)₂-2-(Carboxyethyl-phenyl)pyridine-2-carboxylicacid ester.

14. The method of any one of embodiments 1 to 13, wherein said reactioncomposition comprises further ECL supporting reagents.

15. A method for detecting an analyte in a sample viaelectrochemiluminescence detection, comprising the steps of:

a) incubating the sample with a detection reagent labeled with anelectrochemiluminescent group comprising a transition metal complex, inan embodiment comprising a tris(2,2′-bipyridyl)ruthenium complex(Ru(bpy)₃ ²⁺),

b) separating analyte-bound and analyte-free labeled detection reagent,

c) contacting the separated analyte-bound labeled detection reagent witha branched-chain tertiary amine as specified in any one of embodiments 1to 8 and with an electrode,

d) electrochemically triggering the release of luminescence, and

e) detecting the electrochemiluminescence (ECL) signal thereby detectingthe analyte.

16. A method for detecting an analyte in a sample viaelectrochemiluminescence detection, comprising the steps of:

a) incubating the sample with a detection reagent labeled with anelectrochemiluminescent group comprising a transition metal complex, inan embodiment comprising (Ru(bpy)₃ ²⁺,

b) separating analyte-bound and analyte-free labeled detection reagent,

c) detecting ECL according to the method of any one of embodiments 1 to12, with the detection reagent being the compound comprising atransition metal complex, and

d) detecting the analyte based on the result of the ECL detection instep c).

17. The method according to any one of embodiments 1 to 15,characterized in that the electrode comprises or consists of Au, Jr, Ptor Carbon, in an embodiment is a boron-doped diamond electrode or aglassy carbon electrode; in an embodiment comprises or consist ofplatinum, gold, or glassy carbon, in an embodiment comprises or consistsof platinum.

18. The method according to any of embodiments 15 to 17, characterizedin that detecting said analyte in a sample using ECL is performed in anaqueous solution.

19. The method according to any of embodiments 1 to 18, characterized inthat the method is performed under homogeneous reaction conditions orheterogeneous reaction conditions, in an embodiment under homogeneousreaction conditions, in a further embodiment under heterogeneousreaction conditions.

20. The method according to any of embodiments 1 to 19, characterized inthat the branched-chain tertiary amine is selected from the groupconsisting of N,N-Dipropyl-N-(sec-butyl)amine (DPIBA),N,N-Di(sec-butyl)-propylamine (DIBPA), and N,N-Diisopropyl-N-ethylamine(DIEA) in an embodiment is DPIBA or DIBPA.

21. The method according to any of the embodiments 1 to 20,characterized in that the reagent composition comprises a branched-chaintertiary amine in a concentration of at least 50 mM, in a concentrationof at most 500 mM, in an embodiment in a concentration of 50 mM to 500mM, in a further embodiment 75 mM to 350 mM, in a further embodiment 100mM to 250 mM.

22. The method according to any of the embodiments 1 to 21,characterized in that the reagent mixture in the detection stepcomprises a preservative.

23. The method according to embodiment 22, characterized in that thereagent mixture in the detection step comprises said preservative in aconcentration of 0.1% to 5%.

24. The method according to any of the embodiments 1 to 23,characterized in that the reagent mixture in the detection stepcomprises a detergent and a buffering agent.

25. The method according to any of embodiments 1 to 24, characterized inthat the reagent mixture in the detection step further comprises a saltand/or an anti-foam agent, in an embodiment further comprises acarboxamide, in a further embodiment further comprises, optionallyhalogenated, acetamide, propanamide, or butyramide, in a furtherembodiment further comprises propanamide.

26. A reagent composition for detecting ECL, comprising

i) a branched-chain tertiary amine, in particular a branched-chaintertiary amine of Formula 1, as coreactant, and

ii) a further ECL reagent.

27. The reagent composition according to embodiment 26, wherein saidfurther ECL reagent is an ECL compound comprising a transition metalcomplex, a preservative, a buffer compound, a detergent, an inorganicsalt, in particular a sodium halogenide, an anti-foaming agent, or anycombination thereof; and/or comprising a carboxamide, in a furtherembodiment, optionally halogenated, acetamide, propanamide, orbutyramide, in a further embodiment propanamide.

28. The reagent composition according to embodiment 26 or 27, wherein,said further ECL reagent is an ECL compound comprising a transitionmetal complex.

29. The reagent composition according to any one of embodiments 26 to28, wherein said further ECL reagent is an ECL compound comprising airidium complex.

30. An ECL reaction composition comprising i) at least one ECL compoundcomprising a transition metal complex and ii) at least onebranched-chain tertiary amine as a coreactant.

31. The ECL reaction composition of embodiment 30, further comprising ananalyte.

32. The ECL reaction composition of embodiment 31, further comprising atleast one analyte-specific reagent.

33. The ECL reaction composition of embodiment 32, wherein saidanalyte-specific reagent is covalently bound to said ECL compoundcomprising a transition metal complex.

34. The ECL reaction composition of embodiment 32, wherein said ECLcompound comprising a transition metal complex is covalently bound to anagent specifically binding to said analyte-specific reagent.

35. The ECL reaction composition of embodiment 31, wherein said ECLcompound comprising a transition metal complex is covalently bound to ananalyte or to a structural analog of the analyte.

36. Use of a branched-chain tertiary amine as specified in any one ofembodiments 1 to 8, of a reagent composition according to any one ofembodiments 26 to 29, and/or of an ECL reaction composition according toany one of embodiments 30 to 35 in the detection of ECL.

37. A kit for detecting ECL comprising i) a branched-chain tertiaryamine and ii) an ECL reagent.

38. The kit of embodiment 37, wherein said ECL reagent is an ECLcompound comprising a transition metal complex and/or an ECL supportingreagent.

39. The kit of embodiment 37 or 38, further comprising an electrode, inan embodiment an electrode comprising or consisting of Au, Ir, Pt orCarbon, in an embodiment a boron-doped diamond electrode or a glassycarbon electrode; in a further embodiment an electrode comprising orconsisting of platinum platinum, gold, or a glassy carbon electrode, inan embodiment embedded in a reaction chamber.

40. An ECL device comprising a branched-chain tertiary amine asspecified in any one of embodiments 1 to 8.

EXAMPLE 1: SYNTHESIS/SOURCES OF COMPOUNDS

N,N-Dipropyl-N-(sec-butyl)amine (CAS Registry Number 60021-91-2,“DPIBA”) was synthesized according to Gheorghe, Ruxandra et al.;Chirality, 2008 , Vol. 20, #10, p 1085- 1091, and Takayama et al.;Journal of Organic Chemistry, 1979, Vol. 44, p 2871-2872.

N,N-Di(sec-butyl)-propylamine (CAS Registry Number 1872850-05-9, DIBPA)was synthesized according the following procedure: 100 gdi-sec-butylamine was dissolved in 420 ml methanol and cooled to 0° C.under vigorous stirring. 49.4 g propionaldehyde was dissolved in 82 mlmethanol and slowly added to the solution. 205 g of sodiumtriacetoxyborohydride was added in 5 portions to the cooled solution thesolution was stirred for 2 h at 0° C., afterwards slowly heated up to30° C. and stirred at room temperature for 36 h. The suspension wasfiltrated and the solvent partly removed to ca. 500 ml by evaporation.

500 ml ethyl acetate and 500 ml H₂O were added and the pH adjusted to pH10 by adding a 10 molar NaOH solution. After separation of the organicphase the H₂O phase was extracted another time with 500 ml ethyl acetateand the combined organic phases completely evaporated after drying.

The residue was distilled under high vacuum.

¹H-NMR (500 MHz): 0.85-0.99 ppm, 15 H (—CH₃), 1.21-1.43 ppm, 6 H(—CH₂—), 2.35-2.42 and 2.60-2.66 ppm, 4 H (—N—CH—).

Ethyl-diisopropyl-amine=diisopropylethylamine=CAS Registry Number7087-68-5 (=DIEA) was from Sigma Aldrich.

EXAMPLE 2: VOLTAGE DEPENDENCE OF SPECIFIC AND BACKGROUND SIGNALS

ECL measurements were carried out using a Roche Elecsys® breadboard,which is similar to a Roche Elecsys® 1010 or a Roche Elecsys® 2010comprising the capability to apply different potentials at the workingelectrode. The protocols for the assays mentioned below were used asrecommended for Roche Elecsys® 2010. The dependence of ECL on measuringvoltage was studied using buffers having various compositions. Thegeneral composition of the buffer was as follows:

300 mM Phosphate

0.1% polidocanol

coreactand as specified below; the final pH was adjusted to pH 6.8 usingKOH/H₃PO₄. The coreactands and their concentrations were:

Buffer A (TPA): 180 mM tripropylamine

Buffer B (DPIBA): 120 mM dipropylisobutylamine

Buffer C (DIEA): 200 mM diisopropylethylamine

Buffer D (TBA): 180 mM tributylamine

Buffer E (EP): 200 mM ethylpiperidine

Buffer F (DIBPA): 80mM diisobutylpropylamine

The coreactand concentrations of the buffers reported here are theoptimized concentrations with regard to signal yield. Buffer A comprisesthe buffer composition of ProCell without preservative and served as areference buffer. ProCell is the state of the art buffer currently usedfor Elecsys analyzers. EP, TBA and Phosphate were purchased from SigmaAldrich and were used without further purification.

Buffers A-F were used to determine the ECL of the assay buffer itself(background, FIG. 2 ) and of an artificial immunoassay (SAP, FIG. 1 ).The latter is an assay including RuBpy labeled microparticles for a highspecific signal. Measurements of background and SAP where carried out atdifferent measuring voltages and the ECL signal was recorded. The ratiobetween SAP and background is a good indicator to compare the impact ofthe different buffers on assay sensitivity (FIG. 3 ).

A consistent property of all amines is that their background increasesexponentially with the applied voltage. A fair description for allcurves is given by:

$\begin{matrix}{\frac{S}{S_{\max}} = {0.003\mspace{14mu} e^{\frac{0.0039}{mV}U_{pot}}}} & \left( {{Eq}.\mspace{14mu} 1} \right)\end{matrix}$

A commonly used measuring voltage for TPA is 1400 mV. According to Eq.1,a voltage decrease to 1100 mV decreases the BG by a factor of ˜3.8.However, this decrease is compensated by a strong loss in the specificECL signal. In contrast, the unique voltage dependence of branchedcoreactands allows the utilization of low measuring voltages thusimproving the S/BG ratio significantly.

EXAMPLE 3: ASSAY PERFORMANCE COMPARISON IN TNT ASSAY

As a commercial in vitro diagnostic assay, the Elecsys® TnT assay(TroponinT1.Gen assay for Elecsys®; Order-No.: 05092744-190) was used todetermine the signal to noise ratio. The analyte free sample (DilMa;Order-No.: 3609987) was used in the TnT assay to give the low levelbackground signal (N). TnT calibrator 2 (TnT Cal 2; Order-No.:05092752-190) was used in the TnT assay to give a high signal value (S).

Each assay was carried out using various bead types. These were bead A(standard Elecsys beads used in Elecsys® TnT assay), bead B (M-270,strepatividin coated carboxyl beads, Invitrogen) and Bead C (M-280,streptavidin coated tosyl beads, Invitrogen). To have a reference, theassays were carried out with buffer A under standard Elecsys conditions(1400 mV measuring voltage) using bead types A-C. The signals obtainedserved as a 100% reference for the standard conditions. A second set ofexperiments was carried in the same way but using buffer B and theoptimized conditions according to the results found in example 2 (1100mV measuring voltage) (FIG. 4 ). As can be seen from FIG. 5 , anincrease in S/BG ratio of ˜2 can be achieved independently of the beadtype used.

EXAMPLE 4: VOLTAGE DEPENDENT SIGNAL BEHAVIOR OF IR AND RU LABEL

Moreover, the voltage dependent signal behavior of Ruthenium and Iriduimlabels in SAP assays was analyzed. In one case, the streptavidin coatedmicroparticles used were labeled with Ruthenium-complexes(Biotin-DADOO-Sulfo-Ru, formula (VIII)) and in another case withIridium-complexes (Biotin-DADOO-IB3/47, formula (IX)). Signals werenormalized with the maximum ECL signal to compare the curves. In case ofIr-label, 6% of the specific signal is lost when the measuring voltageis lowered from 1400 mV to 1100 mV. The same decrease in measuringvoltage leads to a 28% signal decrease in the case of Ru-label.

Biotin-DADOO-Sulfo-BPRu:

Biotin-DADOO-IB3/47:

EXAMPLE 5: IMPACT OF ALPHA-BRANCHED SIDE CHAINS ON AMINE OXIDATIONPOTENTIAL

In order to evaluate the effect of side chain branching on tertiaryamine oxidation potentials, cyclic voltammograms (CVs) of TPA, DPIBA andDIBPA in phosphate buffer were recorded. The coreactand solutions wereintroduced into an Elecsys measuring cell V 7.0 which was connected to aSP-300 Potentiostat from Biologic, France. The open circuit potentialwas chosen as a starting potential and the CV was measured at a scanrateof 0.3 V/s between 1.1 and −0.5 V. The obtained CVs were subsequentlyfitted with the fit function available in the potentiostat software(EC-labs) to estimate the standard oxidation potential of the differentcoreactands. Obviously the oxidation potential is reduced from TPA toDPIBA to DIBPA in line with the number of branched side chains of thedifferent coreactands. The reduced oxidation potential of such aminesexplains the reduced potential necessary for ECL generation.

1-15. (canceled)
 16. A reagent composition for detecting ECL, comprisingi) a branched-chain tertiary amine, in particular a branched-chaintertiary amine as specified in claim 1, as coreactant, and ii) a furtherECL reagent wherein said branched-chain tertiary amine is one ofcompounds 1 to 122 of any one of Tables 1 to
 4. 17. The reagentcomposition of claim 16, wherein said further ECL reagent is an ECLcompound comprising a transition metal complex, a preservative, a buffercompound, a detergent, an inorganic salt, in particular a sodiumhalogenide, or an anti-foaming agent, or any combination thereof; and/orcomprising a carboxamide or amide, in a further embodiment furthercomprising, optionally halogenated, acetamide, propanamide, orbutyramide, in a further embodiment further comprising propanamide. 18.The reagent composition of claim 16, wherein said further ECL reagent isan ECL compound comprising a transition metal complex and/or an ECLsupporting reagent.
 19. The reagent composition of claim 16, whereinsaid branched-chain tertiary amine is one of compounds 1 to 83 of anyone of Tables 1 to
 3. 20. The reagent composition of claim 16, whereinsaid branched-chain tertiary amine is one of compounds 1 to 75 of Table1 or
 2. 21. The reagent composition of claim 16, wherein thebranched-chain tertiary amine is a compound according to the generalformula (VII):

wherein R¹⁰ to R¹⁵ are independently selected from —H and methyl,wherein at least one of R¹⁰ to R¹² is methyl; and wherein if R¹⁰, R¹¹and R¹² are methyl, at least one of R¹³ to R¹⁵ is methyl.
 22. Thereagent composition of claim 16, wherein said branched-chain tertiaryamine is N,N-Di(sec-butyl)-propylamine (“N,N-Diisobutyl-propylamine”,DIBPA), N,N-Dipropyl-N-(sec-butyl)amine (“N,N-Dipropyl-isobutylamine”,DPIBA), and/or N,N-Diisopropyl-N-ethylamine (DIEA).
 23. The reagentcomposition of claim 16, wherein said branched-chain tertiary amine isis DIBPA or DPIBA.
 24. The reagent composition of claim 16, wherein saidbranched-chain tertiary amine is DPIBA.
 25. The reagent composition ofclaim 16, wherein said branched-chain tertiary amine is DIPBA.
 26. Thereagent composition of claim 16, comprising said branched-chain tertiaryamine in a concentration of 50 mM to 500 mM.
 27. The reagent compositionaccording to claim 16, wherein said further ECL reagent is an ECLcompound comprising a transition metal complex.
 28. The reagentcomposition according to claim 16, wherein said further ECL reagent isan ECL compound comprising an iridium complex.
 29. A kit for detectingECL comprising i) a branched-chain tertiary amine and ii) an ECLreagent, wherein said branched-chain tertiary amine is one of compounds1 to 122 of any one of Tables 1 to
 4. 30. The kit of claim 29, whereinsaid ECL reagent is an ECL compound comprising a transition metalcomplex and/or an ECL supporting reagent.
 31. The kit of claim 29,further comprising an electrode.
 32. The kit of claim 31, wherein saidelectrode comprises or consists of Au, Ir, Pt or Carbon.
 33. The kit ofclaim 31, wherein said electrode is a boron-doped diamond electrode or aglassy carbon electrode.
 34. The kit of claim 31, wherein said electrodecomprises or consists of platinum, gold, or is a glassy carbonelectrode.
 35. The kit of claim 31, wherein said electrode is embeddedin a reaction chamber.